Therapeutic alteration of transplantable tissues through in situ or ex vivo exposure to rna interference molecules

ABSTRACT

The present invention, at least in part, relates to the discovery of efficacious delivery of an RNAi agent (in preferred aspects of the invention, an siRNA) to a transplantable tissue. Organ rejection, transplantation-mediated transmission of viral infection, and triggering of apoptosis in transplanted tissues can each be minimized by the methods and compositions of the instant invention. The RNAi agent(s) of the instant invention can be delivered as “naked” molecules, or using liposomal and other modes of delivery, to transplantable tissues. Such delivery can occur via perfusion of the RNAi agent in solution through the vasculature of a whole or partial organ; or tissues including transplantable cells and cell lines may be bathed, injected or otherwise treated with RNAi agents. Preferred transplantable tissues include, for example, pancreas, liver, kidney, heart, lung, and all cells and cell lines derived from such tissues (e.g., pancreatic islet cells that may, e.g., be transplanted as a treated population).

RELATED APPLICATIONS

This application is a continuation of U.S. Utility application Ser. No.14/840,187, filed Aug. 31, 2015 (now allowed), which is a continuationof Ser. No. 14/591,073, filed Jan. 7, 2015 (now U.S. Pat. No.9,150,861), which is a continuation of Ser. No. 13/738,353, filed Jan.10, 2013 (now U.S. Pat. No. 8,940,709), which is a continuation of U.S.Utility application Ser. No. 11/179,792, filed Jul. 11, 2005 (now U.S.Pat. No. 8,361,976), which in turn claims the benefit of U.S.Provisional Patent Application Ser. No. 60/586,530, filed Jul. 9, 2004,entitled “Therapeutic Alteration of Transplantable Tissues Through InSitu or Ex Vivo Exposure to RNA Interference Molecules.” The entirecontents of the referenced applications are incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Feb. 27, 2019, is namedSequence_Listing_CRF_101739-000006 and is 4,714 bytes in size.

BACKGROUND OF THE INVENTION

Organ rejection is the process whereby a patient's own immune systemrecognizes the cell surface markers (antigens) within a transplantedorgan as foreign (Joosten et al., 2003, Transpl Int. 2003 16, 137-45).Immunologic recognition of foreign cell surface antigens leads todestruction of the foreign tissue: in the case of transplanted organs,the chronic rejection process destroys the functionality of thetransplant. Class I or class II “major histocompatibility (MHC)” antigenon the surface of the transplanted organ's cells are processed andpresented to the host immune system. Classic initiation of rejectionoccurs when class II molecules are expressed and activate T-helper(CD4⁺) lymphocytes. Cytokines thus released by activated CD4⁺ cells,lead to margination and recognition of foreign class I molecules bycytotoxic T lymphocytes (CD8⁺, CTL) (Buckley, R H. J Allergy ClinImmunol, 2003.111, S733-44). This process has been controlled withcurrent pharmacotherapy to the point of reducing acute rejectionepisodes to around 10% within 1 year after transplantation (Ciancio, G.,et al., Transplantation, 2004 77, 244-51). However, most anti-rejectionmedications are toxic to the patient and given long-term, may induceorgan toxicity (Baran, D. A., et al. Am J Cardiovasc Drugs, 2004 4,21-9) and malignancy (Ganschow, R., et al., J Pediatr GastroenterolNutr, 2004 38, 198-203) in some patients. Tolerance in transplantationis the process by which a transplant is performed and maintained withoutthe need for exogenous drugs to prevent rejection.

Infection also presents a significant obstacle to successfultransplantation. Despite the use of novel antiviral and antibiotictherapeutics, opportunistic infections continue to add to the morbidityand mortality associated with transplantation. Herpesviruses, as anexample, account for serious disease in as many as 50% of transplantrecipients (Aiello, F. B., et al., Mod Pathol, 2004.17, 189-96) and thisleads to poor outcomes for many of the infected patients. These virusesincorporate their genome into the cells of a transplanted graft and assuch, the transplanted organ serves as a reservoir for reactivation andinfection in the transplant recipient. Hepatitis C virus leads tocirrhosis and is the most common cause of liver failure amongst patientsawaiting liver transplantation. Recurrence of the Hepatitis C in thetransplanted liver occurs 100% of the time and leads to graft failure(Neumann. U. P., et al., Transplantation, 2004 77, 226-31) and death innearly 10% of these patients. CMV and Hepatitis C are just two examplesof viruses that cause poor outcomes in transplant patients.

Successful transplantation is also limited by ischemic injury (lack ofoxygen) to the transplanted organ as it is removed from a donor. Isletcell transplantation, as an example, is dependant upon high yield ofviable islets separated from a cadaver pancreas. Islet viability isdiminished as a result of apoptosis (programmed cell death) initiated byinsults such as ischemia induced in the organ procurement process. Thereare current strategies that are being employed in genetically alteringislets to prevent such ischemic loss (Fenjves, E. S., et al.,Transplantation. 2004 77, 13-8). Islets from Balb/c mice have beentransduced with a replication-deficient adenovirus expressing wild-typeSurvivin (pAd-Survivin; G. P. Basadonna. PhD. Thesis of CharlotteAriyan, MD. Yale Univ. 2003). Survivin, first described in some humancancers, is a gene related to bcl-2 or the baculovirus IAP (inhibitor ofapoptosis) gene (Ambrosini, G., C., et al. Nat Med, 1997.3, 917-21), butfunctions independently (Adida, C, et al., Lancet, 1998 351, 882-3).Recombinant expression of Survivin counteracts apoptosis in B lymphocyteprecursors (Ambrosini, G., C., et al. Nat Med, 1997 3, 917-21). Whilenot normally expressed in adult cells, Survivin expression can and doesoccur. Enhanced expression of Survivin has been consistently associatedwith inhibition of apoptosis, in vitro (Kobayashi, K., et al., Proc NatlAcad Sci USA, 1999 96, 1457-62; Tamm, L, et al., Cancer Res, 1998 58,5315-20; Mahotka, C, et al. Cancer Res, 1999 59, 6097-102; Suzuki, A.,et al., Oncogene, 2000×) 19, 1346-53; Islam. A., et al., Med PediatrOncol. 2000 35, 550-3) and in vivo (Grossman. D., et al., J Clin Invest,2001 108, 991-9).

In spite of continuing advances in transplantation technology, seriousmorbidity and mortality are still associated with transplantation.Genetic alteration in many ways can be harnessed to improvetransplantation outcomes; however, genetic alteration of tissue has thusfar been most effectively performed through use of viral intermediaries(such as adenoviral transfection). Such use of viral vectors continuesto present a serious danger to transplant patients; and a safer,efficacious method of preventing rejection of transplanted tissues ishighly desirable.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofefficacious delivery of an RNAi agent (in preferred aspects of theinvention, an siRNA) to a transplantable tissue. Organ rejection,transplantation-mediated transmission of viral infection, and triggeringof apoptosis in transplanted tissues may each be minimized by themethods and compositions of the instant invention. The RNAi agent(s) ofthe instant invention may be delivered as “naked” molecules, or usingliposomal and other modes of delivery, to transplantable tissues. Suchdelivery may occur via perfusion of the RNAi agent in solution throughthe vasculature of a whole or partial organ, or tissues includingtransplantable cells and cell lines may be bathed, injected or otherwisetreated with RNAi agents. Preferred transplantable tissues include, forexample, pancreas, liver, kidney, heart, lung, and all cells and celllines derived from such tissues (e.g., pancreatic islet cells that may,e.g., be transplanted as a treated population).

The present invention therefore provides methods for counteringpotential negative transplantation outcomes, including:

-   -   methods for treating a transplantable tissue with an RNAi agent        in order to prevent immune-mediated rejection of transplanted        tissues    -   methods for treating a transplanted tissue(s) with an RNAi agent        in order to prevent growth or propogation of latent viruses that        may be present in the tissue(s)    -   methods for treating a transplanted tissue(s) with an RNAi agent        in order to prevent apoptosis-induced ischemic injury in the        tissue(s)

The instant invention also features treated transplant tissues derivedby the methods of the instant invention.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the result of efficacious administration of anti-GFP siRNAto islet cells ex vivo. Panel A depicts images for cells that were leftuntreated, while panel B cells were administered anti-GFP siRNA andcells in panel C were treated with non-specific siRNA. All cells weretransfected with adeno-GFP (at an M.O.I. of 500:1 viral PFU/islet)following treatment, and fluorescence was then examined.

FIG. 2 depicts results showing the success of direct organ perfusion(specifically, perfusion of pancreatic islet cells in situ via portalvein administration) with siRNA (red fluorescence, Cy-3 labeled). PanelA shows the effect of administration of “naked” siRNA, while cells inpanel B were administered siRNA packaged into liposomes usingLipofectamine 2000. Cells in panel C were left untreated.

FIG. 3 graphically depicts inhibition of native insulin productionobserved in islet cells isolated from whole pancreas following perfusionwith anti-Insulin 2 siRNA. Assays were performed in triplicate, withpositive standard deviations indicated.

FIG. 4 (A,B,C) shows the efficacy of siRNA delivery and function inisolated islets in vitro. FIG. 4A depicts fluorescent images depictinguntreated control, 50 nM, and 400 nM Cy-3-siRNA transfected samples.Right panel shows islets under red fluorescence. Imagemagnification=200×. FIG. 4B depicts a FACS analysis showing siRNAtransfection efficiency in primary islets after liposomal transfectionwith FITC-labeled Luc-siRNA. Single cell suspensions were made after 48hours in culture. The upper panel is untreated control sample, themiddle panel from 50 nm siRNA and bottom panel from 400 nM siRNA. Thevalue in the upper gate represents percent of FITC-positive cells(Comp-FL-1. Y-axis) versus forward scatter (FSC-H, X-axis) FIG. 4Cdepicts normalized real-time PCR data showing percent Ins2 expressionlevels among the different concentrations of (

) Ins2-siRNA treated islet samples compared to (

) non-specific siRNA control (Luc-siRNA) and (

) untreated control samples. PCR performed in triplicate (n=3, bar graphof average with standard deviations shown. T-test P values shown withasterisks, [*]=P<0.05, [**]=P<0.005.

FIG. 5. (A,B,C) depicts the efficacy of delivery and function of siRNAin vivo. FIG. 5A shows fluorescent images revealing Cy-3 siRNAincorporation into isolated islets after intravenous tail vein injection(100 μg). Left panel shows islets under visible light and right panelunder red fluorescence. Upper panel: Control PBS-treated islets pooledfrom 3 BALB/c mice not receiving siRNA. Middle and Lower panel: Isolatedislets from 3 BALB/c mice receiving Cy3-Luc-siRNA. Isolated islets weredispensed into wells of a 48 well plate, cultured for 16 hours, andobserved under fluorescence. Image magnification=200×. FIG. 5B depictsFACS analysis showing siRNA transfection efficiency in isolated isletsafter i.v. tail vein administration of FITC-labeled Luc-siRNA (100 μg).Single cell suspensions were made after overnight culture. The FACSplots show the upper panel is the untreated control sample and thebottom panel is from the FITC-Luc-siRNA treated islets. The value in theupper gate represents percent of FITC-positive cells (Comp-FL-1. Y-axis)versus forward scatter (FSC-H, X-axis) FIG. 5C, shows quantitativeRT-PCR and demonstrates in vivo siRNA function in pancreatic islets. Bargraph showing normalized real-time PCR data showing the percent Ins2expression levels from (

) islets pooled from control treated, (

) Ins2-siRNA treated, and (

) non-specific siRNA control treated BALB/c mice. Samples were run intriplicate n=3 with standard deviation (SD) and student t-test P valueshown with asterisk, [*]=(P<0.05).

DETAILED DESCRIPTION

The present invention relates, at least in part, to the discovery ofmethods and compositions capable of effectively delivering an RNAi agent(in preferred aspects of the invention, an siRNA) to a transplantabletissue. Administration of RNAi agents by the methods and compositions ofthe instant invention offers a safe, efficacious means of combating avariety of issues that can produce suboptimal transplant outcomes. Suchissues primarily include: organ rejection; reactivation of latent andpersistent viruses in transplanted tissues; and ischemic injury thatinduces apoptotic cell death in the transplanted tissue, as described indetail above. Each of these issues are addressed by the presentinvention through delivery to the transplanted tissue of RNAi agentscapable of inhibiting: 1) production or localization of cell surfaceproteins that trigger immune-mediated rejection (e.g., MHC class II andclass I genes); 2) growth or propogation of viruses (e.g., herpesvirus(e.g., herpes simplex, varicella-zoster virus, Epstein-Barr virus,cytomegalovirus (CMV)), hepatitis C, HIV. JC virus) that might beharbored within the transplanted tissue; and 3) molecular triggers ofapoptosis activated in response to ischemic injury. As shown in theexperimental data of the instant invention, delivery of such RNAi agentscan be achieved by contacting tissues (e.g., cells, cell lines, organsections, whole organs, etc.) directly with “naked” RNAi agents; or inpreferred embodiments, RNAi agents in lipid-mediated delivery vehicles(e.g., liposome) and other art-recognized methods of introducingnucleotide-based agents to cells or tissues are employed. In preferredembodiments of the instant invention, such RNAi agents are administeredor delivered to the transplantation tissue through perfusion, injection,or other contact with the transplant tissue prior to completion of thetransplantation process with delivery of the tissue to a recipientsubject.

In one aspect of the invention, a tissue is prepared for transplantationby exposing the tissue to an RNAi agent capable of downmodulating atrait of allograft rejection, such that the tissue is more suitable fortransplantation as compared to an untreated tissue.

In a related aspect of the instant invention, a tissue is prepared fortransplantation by exposing the tissue to an RNAi agent capable ofdownmodulating a trait of suboptimal allograft transplantation, suchthat the tissue is more suitable for transplantation as compared to anuntreated tissue.

In an additional aspect of the instant invention, a tissue is preparedfor transplantation by exposing the tissue to an RNAi agent capable ofdownmodulating an oncogene, such that the tissue is more suitable fortransplantation as compared to an untreated tissue. In a relatedembodiment, a tissue is prepared for transplantation by exposing thetissue to an RNAi agent capable of modulating a tumor suppressor gene,such that the tissue is more suitable for transplantation as compared toan untreated tissue.

In certain preferred embodiments of the instant invention, the RNAiagent is administered to the tissue through the vasculature of thetissue.

In additional preferred embodiments, the RNAi agent is perfused into thetissue.

In other embodiments of the invention, the RNAi agent is administered tothe tissue through injection into the tissue.

In an additional embodiment of the instant invention, the RNAi agent ispresent in a solution in which the tissue is bathed.

In other embodiments, the transplant tissue is an organ.

In certain embodiments, the tissue is an allograft.

In a preferred embodiment of the invention, the transplant tissue is ofpancreatic origin; and in an additional preferred embodiment, thetransplant tissue comprises β-islet cells.

In specific embodiments of the instant invention, exposure of thetransplant tissue to the RNAi agent is performed in vivo. Alternatively,in other embodiments of the instant invention, exposure to the RNAiagent is performed ex vivo.

In certain embodiments, the RNAi agent is administered in a solutionthat additionally comprises saline.

In specific embodiments, the solution in which the RNAi agent isadministered additionally comprises an immunosuppressant.

In other embodiments, the solution in which the RNAi agent isadministered additionally comprises a cancer treatment.

In specific embodiments, the solution containing the RNAi agentadditionally comprises a leukemia treatment.

In another embodiment, the RNAi agent is an siRNA molecule.

In a specific embodiment, the siRNA molecule is a single stranded siRNA(ss-siRNA) molecule; and in one additional embodiment, the ss-siRNAmolecule is modified such that the ss-siRNA molecule has increased insitu or in vivo stability as compared to an unmodified ss-siRNA molecule(refer to WO04/014933, the entirety of which is incorporated herein byreference; Schwarz, D. S., et al. Mol. Cell, 2002 10, 537-548).

In a separate embodiment, the siRNA molecule is a ds-siRNA molecule.

In one embodiment, the RNAi agent comprises an oligonucleotidecomprising a modification selected from the group consisting of the2′-OH group replaced by a H. alkoxy or OR, halogen, SH, SR, amino (suchas NH2, NHR, NR2), or CN group, wherein R is lower alkyl, alkenyl,alkynyl, or aryl.

In a specific embodiment, the RNAi agent comprises a 2′-O-methyloligonucleotide.

In an additional embodiment, the RNAi agent modulates aglucose-activated insulin response.

In another embodiment, the RNAi agent enhances the fitness of a cell ortissue. In a related embodiment, the cell or tissue comprises a β-isletcell.

In an additional embodiment, the RNAi agent is exposed to the tissuethrough lipid-mediated delivery.

In a related embodiment, the RNAi agent is exposed to the tissue as acomponent of a liposomal preparation.

In another embodiment, the RNAi agent is exposed to the tissue usingelectroporation.

In other embodiments, the RNAi agent is directed against a majorhistocompatibility complex class II (MHC II) gene.

In a related embodiment, the RNAi agent downregulates expression of anMHC II gene.

In another related embodiment, the RNAi agent causes decreasedproduction of an MHC II gene product.

In other related embodiments, the RNAi agent alters cellularlocalization of an MHC II gene product.

In an additional related embodiment, the RNAi agent impedes delivery ofan MHC II gene product to the cell surface.

In another embodiment, the RNAi agent is directed against a majorhistocompatibility complex class I (MHC I) gene.

In a related embodiment, the RNAi agent causes decreased production ofan MHC I gene product.

In another related embodiment, the RNAi agent downregulates expressionof an MHC I gene.

In an additional related embodiment, the RNAi agent alters cellularlocalization of an MHC I gene product.

In another related embodiment, the RNAi agent impedes delivery of an MHCI gene product to the cell surface.

In other embodiments, the RNAi agent modulates expression of a geneinvolved in the apoptotic signaling pathway.

In a related embodiment, the RNAi agent inhibits expression of a genethat initiates apoptosis.

In another embodiment, the RNAi agent inhibits the growth of a virus.

In a related embodiment, the RNAi agent inhibits the replication of avirus.

In another embodiment, the RNAi agent inhibits the expression of a viralgene.

In an additional embodiment, the RNAi agent modulates host genes thatmodulate viral replication.

In a related embodiment, the RNAi agent inhibits host genes that promoteviral replication.

In other embodiments, the virus is selected from the following: herpessimplex virus type I (HSV-1), and herpes simplex virus type 2 (HSV-2),cytomegalovirus (CMV), Epstein-Barr virus (EBV), Human Herpesvirus-6,Human Herpesvirus-7, and BK polyomavirus.

In a specific embodiment, the virus is HSV-1.

In another embodiment, the virus is HSV-2.

In an additional embodiment, the virus is CMV.

In another embodiment, the virus is EBV.

In one embodiment, the virus is hepatitis C.

In an additional embodiment, the virus is HIV.

In one embodiment, the virus is Human Herpesvirus-6.

In an additional embodiment, the virus is Human Herpesvirus-7.

In another embodiment, the virus is BK polyomavirus.

In one embodiment, the RNAi agent acts as an antimicrobial orantibacterial agent.

In other embodiments, the RNAi agent downregulates a gene selected fromthe following: ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB,EBRB2, ETSI, ETSI, ETV6, FGR, FOS. FYN, HCR, HRAS, JUN, KRAS, LCK, LYN,MDM2. MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM I, PML, RET, SRC, TALI,TCL3, and YES.

In another aspect of the instant invention, diabetes is treated in atransplanted tissue by exposing an RNAi agent that targets genesinvolved in diabetes to the tissue to be transplanted.

In additional embodiment of the instant invention, more than one RNAiagent is administered to the transplanted tissue.

In certain embodiments, treatment with RNAi agent is performed on avertebrate animal tissue.

In one embodiment, treatment with RNAi agent is performed on a mammaliantissue.

In another embodiment, treatment with RNAi agent is performed on anon-human mammalian tissue.

In an additional embodiment, treatment with RNAi agent is performed on apig or porcine tissue.

In a specific embodiment, treatment with RNAi agent is performed on amouse or mouse tissue.

In another embodiment, treatment with RNAi agent is performed on amonkey or monkey tissue.

In a related embodiment, treatment with RNAi agent is performed on ananimal or tissue therefrom selected from the following: baboon,chimpanzee, and orangutan.

In another embodiment, treatment with RNAi agent is performed on ahuman.

In an additional embodiment, treatment with RNAi agent is performed on ahuman tissue.

In another aspect of the instant invention, tissue prepared by themethods of the instant invention is claimed.

In an additional embodiment of the instant invention, a composition fortreating allograft rejection comprising an RNAi agent that targets amajor histocompatibility class II gene is claimed.

A particular aspect of the invention relates, at least in part, to useof RNA interference as mediated by short-interfering RNA (siRNA) as anon-viral means to silence genes in tissue. Little data exists aboutgene therapy using siRNA in pancreas tissue (or in many other tissues).To determine if siRNA treatment can silence an endogenous gene inpancreatic islets, a murine model was developed using the endocrinepancreas. In this model, the Insulin 2 (Ins2) gene was targeted withsiRNA and quantitative RT-PCR, fluorescent microscopy, and FACS wereused to measure transcript levels and siRNA cellular uptake andtransfection efficiency. Isolated pancreatic islets were transfectedwith siRNA in vitro using a liposomal delivery method in a dosetitration (50-400 nM) or pooled from BALB/c mice having received siRNA(100 g) via hydrodynamic tail vein injection. In these assays, the Ins2transcript level was significantly reduced by 55% in vitro, with FACSdata showing a transfection efficiency over 45% with the 400 nMconcentration. In vive delivery of siRNA to pancreatic islets revealed a33% reduction in Ins2 mRNA levels, while siRNA was able to be detectedin 19% of isolated islet cells. Thus, RNA interference was successfullyused to silence an endogenous, tissue specific gene (Ins2) in pancreaticislets when transfected in vitro or administered in vivo.

So that the invention may be more readily understood, certain terms arefirst defined.

The term “a trait of allograft rejection”, as used herein, refers to anycharacteristic or activity, process or pathway involved in producing, orsymptomatic of, immune-mediated rejection of a transplanted tissue.

The term “a trait of suboptimal allograft transplantation”, as usedherein, refers to any characteristic or activity, process or pathwayinvolved in, or symptomatic of, creating any dangerous, toxic, orotherwise problematic outcome during tissue transplantation. Inpreferred embodiments of the instant invention, such traits include,e.g., immune-mediated rejection, transmission of growth- orpropogation-competent virus present in the transplanted tissue, andinduction of apoptosis mediated by ischemic injury to the transplantedtissue.

As used herein, the term “oncogene” refers to a gene, the aberrantexpression or activity of which stimulates cell growth (e.g., abnormalcell growth). When the level of expression or activity of an oncogene ina cell is reduced, the rate of cell growth can be reduced or the cellmade quiescent. In the context of the present invention, oncogenesinclude intracellular proteins, as well as extracellular growth factorswhich may stimulate cell proliferation through autocrine or paracrinefunction. Examples of human oncogenes against which RNAi agents candesigned include c-myc, c-myb, mdm2, PKA-I (protein kinase A type 1),Abl-1, Bcl2, Ras, c-Raf kinase, CDC25 phosphatases, cyclins, cyclindependent kinases (cdks), telomerase, PDGF/sis, erb-B, fos, jun, mos,and src, to name but a few. In the context of the present invention,oncogenes also include a fusion gene resulted from chromosomaltranslocation, for example, the Bcr/Abl fusion oncogene.

The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refersto a polymer of ribonucleotides. The term “DNA” or “DNA molecule” ordeoxyribonucleic acid molecule” refers to a polymer ofdeoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., byDNA replication or transcription of DNA, respectively). RNA can bepost-transcriptionally modified. DNA and RNA can also be chemicallysynthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA,respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA anddsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNAthat specifies the amino acid sequence of one or more polypeptidechains. This information is translated during protein synthesis whenribosomes bind to the mRNA.

The term “nucleoside” refers to a molecule having a purine or pyrimidinebase covalently linked to a ribose or deoxyribose sugar. Exemplarynucleosides include adenosine, guanosine, cytidine, uridine andthymidine. The term “nucleotide” refers to a nucleoside having one ormore phosphate groups joined in ester linkages to the sugar moiety.Exemplary nucleotides include nucleoside monophosphates, diphosphatesand triphosphates. The terms “polynucleotide” and “nucleic acidmolecule” are used interchangeably herein and refer to a polymer ofnucleotides joined together by a phosphodiester linkage between 5′ and3′ carbon atoms.

The term “RNA interference” or “RNAi” (also referred to in the art as“gene silencing” and/or “target silencing”, e.g., “target mRNAsilencing”), as used herein, refers generally to a sequence-specific orselective process by which a target molecule (e.g., a target gene,protein or RNA) is downregulated. In specific embodiments, the processof “RNA interference” or “RNAi” features degradation of RNA molecules,e.g., RNA molecules within a cell, said degradation being triggered byan RNAi agent. Degradation is catalyzed by an enzymatic, RNA-inducedsilencing complex (RISC). RNAi occurs in cells naturally to removeforeign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragmentscleaved from free dsRNA which direct the degradative mechanism to othersimilar RNA sequences. Alternatively, RNAi can be initiated by the handof man, for example, to silence the expression of target genes.

The term “RNAi agent”, as used herein, refers to an RNA (or analogthereof), having sufficient sequence complementarity to a target RNA(i.e., the RNA being degraded) to direct RNAi. An RNAi agent having a“sequence sufficiently complementary to a target RNA sequence to directRNAi” means that the RNAi agent has a sequence sufficient to trigger thedestruction of the target RNA by the RNAi machinery (e.g., the RISC) orprocess. An RNAi agent having a “sequence sufficiently complementary toa target RNA sequence to direct RNAi” is also intended to mean that theRNAi agent has a sequence sufficient to trigger the translationalinhibition of the target RNA by the RNAi machinery or process.

As used herein, the term “small interfering RNA” (“siRNA”) (alsoreferred to in the art as “short interfering RNAs”) refers to an RNA (orRNA analog) comprising between about 10-50 nucleotides (or nucleotideanalogs) which is capable of directing or mediating RNA interference.Preferably, an siRNA comprises between about 15-30 nucleotides ornucleotide analogs, more preferably between about 16-25 nucleotides (ornucleotide analogs), even more preferably between about 18-23nucleotides (or nucleotide analogs), and even more preferably betweenabout 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22nucleotides or nucleotide analogs).

As used herein, the term “microRNA” (“miRNA”) refers to an RNA (or RNAanalog) comprising the product of an endogenous, non-coding gene whoseprecursor RNA transcripts can form small stem-loops from which maturemiRNAs are cleaved by Dicer (Lagos-Quintana et al., 2001; Lau et al.,2001; Lee and Ambros, 2001; Lagos-Quintana et al., 2002; Mourelatos etal., 2002; Reinhart et al., 2002; Ambros et al., 2003; Brennecke et al.,2003; Lagos-Quintana et al., 2003; Lim et al., 2003a; Lim et al.,2003b). miRNAs are encoded in genes distinct from the mRNAs whoseexpression they control. Mature miRNAs represent the single strandedproduct of Dicer cleavage that then function as guide RNA fragments whenincorporated into the RISC complex.

As used herein, the term “antisense strand” of an siRNA or RNAi agentrefers to a strand that is substantially complementary to a section ofabout 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22nucleotides of the mRNA of the gene targeted for silencing. Theantisense strand has sequence sufficiently complementary to the desiredtarget mRNA sequence to direct target-specific RNA interference (RNAi),e.g., complementarity sufficient to trigger the destruction of thedesired target mRNA by the RNAi machinery or process. The term “sensestrand” of an siRNA or RNAi agent refers to a strand that iscomplementary to the antisense strand. Antisense and sense strands canalso be referred to as first or second strands, the first or secondstrand having complementarity to the target sequence and the respectivesecond or first strand having complementarity to said first or secondstrand.

As used herein, the term “guide strand” refers to a strand of an RNAiagent, e.g., an antisense strand of an siRNA duplex, that enters intothe RISC complex and directs cleavage of the target mRNA. The term“guide strand” is often used interchangeably with the term “antisensestrand” in the art.

A “target gene” is a gene whose expression is to be selectivelyinhibited or “silenced.” This silencing is achieved by cleaving the mRNAof the target gene by an RNAi pathway or process.

As used herein, the term or “MHC molecule” means an MHC class I moleculeand/or an MHC class II molecule.

As used herein, the term “MHC class I” or “MHC I” refers to the humanMajor Histocompatibility Complex class I molecules, binding peptides orgenes. The human MHC region, also referred to as HLA, is found onchromosome six and includes the class I region and the class II region.Within the MHC class I region are found the HLA-A, HLA-B or HLA-Csubregions for class I α chain genes. The human gene forβ₂-microglobulin is located outside the MHC complex on a separatechromosome. As used herein, the term “MHC class I molecule” means acomplex of an MHC class I α chain and a β₂-microglobulin chain. MHCclass I molecules normally bind peptides which are generated in thecytosol and transported to the endoplasmic reticulum. After bindingthese peptides, the class I MHC-peptide complex is presented on the cellsurface where it may be recognized by T cells. The majority of boundpeptides have a length of 8-10 amino acids, although they may be as long16 or as short as 2 (Udaka et al., (1993) Proc. Natl. Acad. of Sci.(USAt 90:11272-11276). See, generally, Roitt et al., eds. Immunology(1989) Gower Medical Publishing, London.

As used herein, the term “MHC class 11” or “MHC 11” refers to the humanMajor Histocompatibility Complex class II molecules, binding peptides orgenes. The human MHC region, also referred to as HLA, is found onchromosome six and includes the class I region and the class II region.Within the MHC class 11 region are found the DP, DQ and DR subregionsfor class II α chain and β chain genes (i.e., DPα, DPβ, DQα, DQβ, DRα,and DRβ). As used herein, the term “MHC class II molecule” means acomplex of an MHC class II α chain and an MHC class II β chain. MHCclass II molecules normally bind peptides in an intracellular processingcompartment and present these peptides on the surface of antigenpresenting cells to T cells. The majority of bound peptides have alength of 13-18 amino acids but it is the peptide side chains of anapproximately 9 amino acid core segment that occupy pockets of the MHCclass II binding cleft and determine the specificity of binding (Brownet al., (1993) Nature 364:33-39; Stem et al., (1994) Nature368:215-221). See, generally, Roitt et al., eds. Immunology (1989) GowerMedical Publishing, London.

The term “oligonucleotide” refers to a short polymer of nucleotidesand/or nucleotide analogs. The term “RNA analog” refers to anpolynucleotide (e.g., a chemically synthesized polynucleotide) having atleast one altered or modified nucleotide as compared to a correspondingunaltered or unmodified RNA but retaining the same or similar nature orfunction as the corresponding unaltered or unmodified RNA. As discussedabove, the oligonucleotides may be linked with linkages which result ina lower rate of hydrolysis of the RNA analog as compared to an RNAmolecule with phosphodiester linkages. For example, the nucleotides ofthe analog may comprise methylenediol, ethylene diol, oxymethylthio,oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phophoroamidate,and/or phosphorothioate linkages. Preferred RNA analogues include sugar-and/or backbone-modified ribonucleotides and/or deoxyribonucleotides.Such alterations or modifications can further include addition ofnon-nucleotide material, such as to the end(s) of the RNA or internally(at one or more nucleotides of the RNA). An RNA analog need only besufficiently similar to natural RNA that it has the ability to mediate(mediates) RNA interference.

As used herein, the “5′ end”, as in the 5′ end of an antisense strand,refers to the 5′ terminal nucleotides, e.g., between one and about 5nucleotides at the 5′ terminus of the antisense strand. As used herein,the “3′ end”, as in the 3′ end of a sense strand, refers to the region,e.g., a region of between one and about 5 nucleotides, that iscomplementary to the nucleotides of the 5′ end of the complementaryantisense strand.

As used herein, the term “isolated RNA” (e.g., “isolated siRNA” or“isolated RNAi agent”) refers to RNA molecules which are substantiallyfree of other cellular material, or culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized.

As used herein, “modulation” may constitute any alteration at any pointin time of the relative activity or abundance of, for example, a gene,gene product, or pathway, as compared to wild-type levels. (Examples ofsuch modulation include: gene knockouts, transgenic expression of a geneor mutant form of a gene, expression of a mutant form of a native gene,underexpression and overexpression of a gene.) An “RNAi modulatorycompound” is therefore any compound capable of modulation in any mannerof RNAi.

As used herein, a “reduced activity” is one that is at least 5% lessthan that of the polypeptide or protein encoded by the wild-type nucleicacid molecule or gene, preferably at least 5-10% less, more preferablyat least 10-25% less and even more preferably at least 25-50%, 50-75% or75-100% less than that of the polypeptide or protein encoded by thewild-type nucleic acid molecule or gene. Ranges intermediate to theabove-recited values, e.g., 75-85%, 85-90%, 90-95%, are also intended tobe encompassed by the present invention. As used herein, a “reducedactivity” also includes an activity that has been deleted or “knockedout” (e.g., approximately 100% less activity than that of thepolypeptide or protein encoded by the wild-type nucleic acid molecule orgene). Likewise, an “enhanced activity” is one that is at least 5%greater than that of the polypeptide or protein encoded by the wild-typenucleic acid molecule or gene, preferably at least 5-10% greater, morepreferably at least 10-25% greater and even more preferably at least25-50%, 50-75% or 75-100% greater or 100% or more greater (two-fold orgreater elevated) than that of the polypeptide or protein encoded by thewild-type nucleic acid molecule or gene. Ranges intermediate to theabove-recited values, e.g., 75-85%, 85-90%, 90-95%, are also intended tobe encompassed by the present invention.

Activity can be determined according to any well accepted assay formeasuring activity of a particular protein of interest. Activity can bemeasured or assayed directly, for example, by measuring an activity of aprotein isolated or purified from a cell. Alternatively, an activity canbe measured or assayed within a cell or in an extracellular medium or ina crude extract of cells. Additionally, activity of a targeted proteinmay also be measured in a whole organism.

The term “tissue”, as used herein, refers to any biological entityderived from an organism (directly or via an isolated progenitor cell orpopulation) that is comprised of cells, including whole organs, organsections and subsections, tumor cells, cells, cell lines, etc. The termalso includes plant cells, as used herein.

The term “perfusion”, as used herein, refers to the act of pouring overor through, especially the passage of a fluid through the vessels of aspecific organ. In specific embodiments of the instant invention, fluidscontaining RNAi agents are perfused through the vasculature oftransplant tissues.

The terms “apoptosis” or “programmed cell death,” refers to thephysiological process by which unwanted or useless cells are eliminatedduring development and other normal biological processes. Apoptosis, isa mode of cell death that occurs under normal physiological conditionsand the cell is an active participant in its own demise (“cellularsuicide”). It is most often found during normal cell turnover and tissuehomeostasis, embryogenesis, induction and maintenance of immunetolerance, development of the nervous system and endocrine-dependenttissue atrophy. Apoptosis may also be triggered by external events andstimuli, such as ischemic injury in the case of certain preferredembodiments of the instant invention. Cells undergoing apoptosis showcharacteristic morphological and biochemical features. These featuresinclude chromatin aggregation, nuclear and cytoplasmic condensation,partition of cytoplasm and nucleus into membrane-bound vesicles(apoptotic bodies) which contain ribosomes, morphologically intactmitochondria and nuclear material. In vivo, these apoptotic bodies arerapidly recognized and phagocytized by either macrophages or adjacentepithelial cells. Due to this efficient mechanism for the removal ofapoptotic cells in vivo no inflammatory response is elicited. In vitro,the apoptotic bodies as well as the remaining cell fragments ultimatelyswell and finally lyse. This terminal phase of in vitro cell death hasbeen termed “secondary necrosis.”

“Inhibition of gene expression” refers to the absence (or observabledecrease) in the level of protein and/or mRNA product from a targetgene. “Specificity” refers to the ability to inhibit the target genewithout manifest effects on other genes of the cell. The consequences ofinhibition can be confirmed by examination of the outward properties ofthe cell or organism or by biochemical techniques such as RNA solutionhybridization, nuclease protection. Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radiohnmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS).

The term “glucose-activated insulin response”, as used herein, refers tothe process by which the islet cells of the pancreas sense and respondto glucose levels (e.g., circulating blood glucose levels for isletcells in vivo). High circulating blood glucose levels will typicallytrigger elevated production and secretion of insulin by pancreatic isletcells in vivo, through molecular signaling mechanisms that are widelyrecognized in the art. These glucose signaling responses may bemodulated by RNAi agents (e.g., shRNA, miRNA, siRNA, etc.; Katavama, K,et al. FEBS Lett. 2004 560, 178-82; Huang, A., et al FEBS Lett. 2004558, 69-73).

The term “loss-of-function,” as it refers to genes inhibited by thesubject RNAi method, refers a diminishment in the level of expression ofa gene when compared to the level in the absence of RNAi agents.

A gene “involved” in a disorder includes a gene, the normal or aberrantexpression or function of which effects or causes a disease or disorderor at least one symptom of said disease or disorder.

Various methodologies of the instant invention include a step thatinvolves comparing a value, level, feature, characteristic, property,etc. to a “suitable control”, referred to interchangeably herein as an“appropriate control”. A “suitable control” or “appropriate control” isany control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc. determined prior to performing an RNAi methodology, asdescribed herein. For example, a transcription rate, mRNA level,translation rate, protein level, biological activity, cellularcharacteristic or property, genotype, phenotype, etc. can be determinedprior to introducing an RNAi agent of the invention into a cell ororganism. In another embodiment, a “suitable control” or “appropriatecontrol” is a value, level, feature, characteristic, property, etc.determined in a cell or organism, e.g., a control or normal cell ororganism, exhibiting, for example, normal traits. In yet anotherembodiment, a “suitable control” or “appropriate control” is apredefined value, level, feature, characteristic, property, etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. The RNAi Pathway

In recent years, numerous studies have shed light on the mechanisms ofRNA-mediated gene silencing. Since the discoveries ofpost-transcriptional gene silencing (PTGS) in plants and RNAinterference (RNAi) in C. elegans, RNA-mediated silencing mechanismshave been identified in a variety of organisms, indicating that thisgene silencing phenomenon is evolutionarily conserved. In PTGS or RNAi,the presence of double-stranded RNA (dsRNA) leads to thesequence-specific degradation of corresponding mRNA, silencingexpression of a target gene having homology with the dsRNA (Hannon, G.J. Nature, 2002 418: 244-51; Hutvagaer and Zamore Curr Opin Genet Dev,2002 12: 225-32). Studies in plants indicate that PTGS likely evolved asa primitive defense system against molecular parasites, such as virusesand transposons. As a result, there are many examples of viral factorsfrom plant viruses that have been found to inhibit PTGS (Waterhonse, P.M., et al. Nature, 2001 411: 834-42; Voinnet, O. Trends Genet, 2001 17:449-59). Recently, a gene product that interferes with RNAi wasidentified in an insect virus (Li, H., et al. Science. 2002 296:1319-21).

The RNAi pathway is the best studied of the RNA-mediated gene silencingmechanisms. In the RNAi response, the RNase III enzyme, Dicer, cleavesdouble-stranded RNA (dsRNA) to produce small interfering RNAs (siRNAs).The siRNAs are 21-23 nucleotide dsRNAs with specialized features, suchas a 2 base 3′ overhang, 5′ phosphate groups, and 3′ hydroxyl groups.Acting as functional intermediates in the pathway, siRNAs areincorporated into a large enzyme complex called the RNA-inducedsilencing complex (RISC). Here, the siRNA functions as a guide to targetthe specific cleavage of mRNA, presumably by an enzyme within thecomplex. In plants and invertebrates, long dsRNAs can be expressed orintroduced to initiate specific gene silencing (Hannon, G. J. Nature,2002 418: 244-51; Hutvagaer and Zamore Curr Opin Genet Dev, 2002 12:225-32). However, in mammalian cells, the introduction of long (>40 bp)dsRNA can lead to the sequence-independent activation of the interferon(IFN) response, which inhibits translation in the cell (Samuel, C. E.Clin Microbiol Rev, 2001 14, 778-809). In order to bypass the IFNresponse, siRNAs can be directly administered to cells and the desiredgene-silencing effect is still achieved (Zamore, P. D., et al. Cell,2000 101, 25-33; Elbashir, S. M., et al. Nature, 2001 411, 494-8).

In addition to cleavage of long dsRNAs, Dicer also plays an importantrole in development by processing micro RNAs (miRNA) from short stemloop precursors. These miRNAs have been implicated in the control ofgene expression during specific stages of development. Unlike theprocessing of dsRNA to double-stranded siRNAs, Dicer cleaves the miRNAprecursors to produce single-stranded RNA (ssRNA) molecules of 21-22nucleotides [Hutvagaer and Zamore Curr Opin Genet Dev, 2002 12: 225-32;McManus, M. T. and P. A. Sharp, Nat Rev Genet, 2002 3, 737-47). Theearliest identified miRNAs, lin-4 and let-7, were found to exert theircontrol of gene expression by affecting the translation of mRNA. UnlikesiRNAs, these miRNA did not have perfect or near-perfect (>95%)complementarity with their target RNAs. Rather than specificallydegrading the mRNA, these miRNAs bound to non-identical 21-22 basesequences in the 3′ untranslated region (UTR) of mRNA, therebypreventing translation (Grishok, A., et al., Cell, 2001 106, 23-34).Using bioinformatics approaches, several groups have found numerousexamples of miRNAs in several different organisms (Lau, N. C., et al.,Science, 2001 294, 858-62; Lagos-Quintana, M., et al. Science, 2001 294,853-8; Rhoades, M. W., et al., Cell, 2002.110, 513-20). Intriguingly, arecent study found that a single-stranded miRNA was able to function asan siRNA, specifically degrading target sequences that bore perfectcomplementarity to the miRNA sequence. This study provided evidence thatthe degree of complementarity between a miRNA and its target sequencedetermined whether the miRNA would act as a translational repressor oras a guide in a RISC-like complex leading to degradation of mRNA(Hutvagner, G. and P. D. Zamore. Science, 2002 297, 2056-60). This invitro finding was supported by new reports in plants, where miRNAs withperfect sequence complementarity to targets were found to specificallydegrade these mRNAs (Llave, C, et al., Science, 2002 297, 2053-6). Theapplication of the RNA-mediated silencing mechanisms has revolutionizedgenetic and functional studies of specific genes. Researchers can simplyintroduce long dsRNA or siRNA (depending on the species under study) tosilence the expression of that gene. In mammalian systems, this has beenaccomplished in tissue culture settings by transfecting orelectroporating siRNAs into cells (Elbashir, S. M., et al. Nature, 2001411, 494-8; Jacque, J. M., et al. Nature, 2002 418, 435-8).Additionally, in vivo studies in mice have been carried out by injectingsiRNAs into the animals (McCaffrey, A. P., et al., Nature, 2002 418,38-9). RNAi is thus a powerful method for the study of gene function inanimals and plants and is being developed as a therapy for treatinggenetic disorders and viral infections.

II. RNA Molecules and Agents

The present invention features “small interfering RNA molecules” (“siRNAmolecules” or “siRNA”) and methods (e.g., therapeutic methods) for usingsaid siRNA molecules. An siRNA molecule of the instant invention ispreferably a duplex consisting of a sense strand and complementaryantisense strand, the antisense strand having sufficient complementarityto a target mRNA to mediate RNAi. Because only the antisense strand ofan siRNA duplex incorporates into the RISC to mediate cleavage orsilencing of the target mRNA, a single antisense strand capable ofactivating RISC (e.g., a stable form of the antisense strand) couldmimic the functionality of the siRNA duplex. Preferably, the strands ofan siRNA duplex are aligned such that there are at least 1, 2, or 3bases at the end of the strands which do not align (i.e., for which nocomplementary bases occur in the opposing strand) such that an overhangof 1, 2 or 3 residues occurs at one or both ends of the duplex whenstrands are annealed. Preferably, the siRNA molecule has a length fromabout 10-50 or more nucleotides, i.e., each strand comprises 10-50nucleotides (or nucleotide analogs). More preferably, the siRNA moleculehas a length from about 15-45 or 15-30 nucleotides. Even morepreferably, the siRNA molecule has a length from about 16-25 or 18-23nucleotides. The siRNA molecules of the invention further have asequence that is “sufficiently complementary” to a target mRNA sequenceto direct target-specific RNA interference (RNAi), as defined herein,i.e., the siRNA has a sequence sufficient to trigger the destruction ofthe target mRNA by the RNAi machinery or process.

siRNAs function as the specificity determinants of the RNAi pathway,where they act as guides to direct endonucleolytic cleavage of theirtarget RNAs (Hamilton and Baulcombe. 1999; Hammond et al., 2000; Zamoreet al., 2000; Elbashir et al., 2001b). The two strands of an siRNA arenot equally eligible for assembly into RISC (Schwarz et al., 2003).Rather, both the absolute and relative stabilities of the base pairs atthe 5′ ends of the two siRNA strands determine the degree to which eachstrand participates in the RNAi pathway, siRNA duplexes can befunctionally asymmetric, with only one of the two strands able totrigger RNAi. Asymmetry is also the hallmark of a related class ofsmall, single-stranded, non-coding RNAs, microRNAs (miRNAs).

In general, siRNA containing nucleotide sequences sufficiently identicalto a portion of the target gene to effect RISC-mediated cleavage of thetarget gene are preferred. 100% sequence identity between the siRNA andthe target gene is not required to practice the present invention. Theinvention can tolerate sequence variations within the methods, tissuesand compositions of the invention in order to enhance efficiency andspecificity of RNAi. For example, siRNA sequences with insertions,deletions, and single point mutations relative to the target sequencecan also be effective for inhibition. Alternatively, siRNA sequenceswith nucleotide analog substitutions or insertions can be effective forinhibition.

The siRNAs of the invention can comprise 16-30, e.g., 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in eachstrand, wherein one of the strands is substantially complementary, e.g.,at least 80% complementary (or more, e.g., 85%, 90%, 95%, or 100%) (forexample, having 3, 2, 1, or 0 mismatched nucleotide(s)), to a targetregion. A target region differs by at least one base pair between thewild type and mutant allele, e.g., a target region comprising again-of-function mutation, and the other strand is identical orsubstantially identical to the first strand. The dsRNA molecules of theinvention can be chemically synthesized or can be transcribed be invitro from a DNA template or engineered RNA precursor.

The dsRNA molecules can be designed using any method known in the art,for instance, by using the following protocol:

-   -   1. Beginning with an AUG start codon, search for AA dinucleotide        sequences; each AA and the 3′ adjacent 16 or more nucleotides        are potential siRNA targets. The siRNA should be specific for a        target region that differs by at least one base pair between the        wild type and mutant allele, e.g., a target region comprising        the gain-of-function mutation. In cases where the        gain-of-function mutation is associated with one or more other        mutations in the same gene, the siRNA can be targeted to any of        the mutations. In some cases, the siRNA is targeted to an        allelic region that does not comprise a known mutation but does        comprise an allelic variation of the wild-type (reference)        sequence. The first strand should be complementary to this        sequence, and the other strand is identical or substantially        identical to the first strand. In one embodiment, the nucleic        acid molecules are selected from a region of the target allele        sequence beginning at least 50 to 100 nt downstream of the start        codon, e.g., of the sequence of Insulin 2. Further, siRNAs with        lower G/C content (35-55%) may be more active than those with        G/C content higher than 55%. Thus in one embodiment, the        invention includes nucleic acid molecules having 35-55% G/C        content. In addition, the strands of the siRNA can be paired in        such a way as to have a 3′ overhang of 1 to 4, e.g., 2,        nucleotides. Thus in another embodiment, the nucleic acid        molecules can have a 3′ overhang of 2 nucleotides, such as TT.        The overhanging nucleotides can be either RNA or DNA.    -   2. Using any method known in the art, compare the potential        targets to the appropriate genome database (human, mouse, rat,        etc.) and eliminate from consideration any target sequences with        significant homology to other coding sequences. One such method        for such sequence homology searches is known as BLAST, which is        available at www.ncbi.nlm.nih.gov/BLAST.    -   3. Select one or more sequences that meet your criteria for        evaluation. Further general information about the design and use        of siRNA may be found in “The siRNA User Guide.” available at        http://www.mpibpc.gwdg.de/abteilungen/100/105/sima.html. The        siRNAs of the invention may have one or more modified bases in        the antisense strand, e.g., U(5Br), U(51), and/or DAP. Such        modified siRNAs can be synthesized with the modified base.

Negative control siRNAs should have the same nucleotide composition asthe selected siRNA, but without significant sequence complementarity tothe appropriate genome. Such negative controls can be designed byrandomly scrambling the nucleotide sequence of the selected siRNA; ahomology search can be performed to ensure that the negative controllacks homology to any other gene in the appropriate genome. In addition,negative control siRNAs can be designed by introducing one or more basemismatches into the sequence.

Sequence identity may be determined by sequence comparison and alignmentalgorithms known in the art. To determine the percent identity of twonucleic acid sequences (or of two amino acid sequences), the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100), optionally penalizing the score for the number of gapsintroduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. For certain applications, the alignment can be generated overa certain portion of the sequence aligned having sufficient identity butnot over portions having low degree of identity (i.e., a localalignment). A preferred, non-limiting example of a local alignmentalgorithm utilized for the comparison of sequences is the algorithm ofKarlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68,modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA90:5873-77. Such an algorithm is incorporated into the BLAST programs(version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.

The alignment may also be optimized by introducing appropriate gaps andpercent identity is determined over the length of the aligned sequences(i.e., a gapped alignment). To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, thealignment is optimized by introducing appropriate gaps and percentidentity is determined over the entire length of the sequences aligned(i.e., a global alignment). A preferred, non-limiting example of amathematical algorithm utilized for the global comparison of sequencesis the algorithm of Myers and Miller, CABIOS (1989). Such an algorithmis incorporated into the ALIGN program (version 2.0) which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

Greater than 80% sequence identity, e.g., 80%, 81%, 82%. 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% oreven 100% sequence identity, between the siRNA antisense strand and theportion of the target gene is preferred. Alternatively, the siRNA may bedefined functionally as a nucleotide sequence (or oligonucleotidesequence) that is capable of hybridizing with a portion of the targetgene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C. or 70° C. hybridization for 12-16 hours; followed by washing).Additional preferred hybridization conditions include hybridization at70° C. in 1×SSC or 50° C. in 1×SSC, 50% o formamide followed by washingat 70° C. in 0.3×SSC or hybridization at 70° C. in 4×SSC or 50° C. in4×SSC, 50% formamide followed by washing at 67° C. in 1×SSC. Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(°C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49base pairs in length, Tm(° C.)=81.5+16.6 (log 10[Na+])+0.41 (%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] isthe concentration of sodium ions in the hybridization buffer ([Na+] for1×SSC=0.165 M). Additional examples of stringency conditions forpolynucleotide hybridization are provided in Sambrook, J., E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters9 and 11, and Current Protocols in Molecular Biology, 1995, F. M.Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4, incorporated herein by reference. The length of the identicalnucleotide sequences may be at least about 10, 12, 15, 17, 20, 22, 25,27, 30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.

The RNA molecules of the present invention can be modified to improvestability in serum or in medium for cell and/or organ cultures. In orderto enhance the stability, the 3′-residues may be stabilized againstdegradation, e.g., they may be selected such that they consist of purinenucleotides, particularly adenosine or guanosine nucleotides.Alternatively, substitution of pyrimidine nucleotides by modifiedanalogues, e.g., substitution of uridine by 2′-deoxythymidine istolerated and does not affect the efficiency of RNA interference.

In a preferred aspect, the invention features small interfering RNAs(siRNAs) that include a sense strand and an antisense strand, whereinthe antisense strand has a sequence sufficiently complementary to atarget mRNA sequence to direct target-specific RNA interference (RNAi)and wherein the sense strand and/or antisense strand is modified by thesubstitution of internal nucleotides with modified nucleotides, suchthat in vivo stability is enhanced as compared to a correspondingunmodified siRNA.

RNA molecules of the invention may additionally contain at least onemodified nucleotide analogue. The nucleotide analogues may be located atpositions where the target-specific activity, e.g., the RNAi mediatingactivity is not substantially effected, e.g., in a region at the 5′-endand/or the 3′-end of the RNA molecule. Particularly, the ends may bestabilized by incorporating modified nucleotide analogues.

Preferred nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In preferred backbone-modified ribonucleotides the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In preferred sugar-modifiedribonucleotides, the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR NR₂ or ON, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I. Exemplary art-recognizedmodifications of RNAi agents include, e.g., 2-Fluoro and 2-Chloromodifications and other stabilizing modifications, such as 2′-O-Memodifications and locked nucleic acids (LNA).

Also preferred are nucleobase-modified ribonucleotides, i.e.,ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

Additional modified and conjugated forms of RNAi agents may also be usedin the methods of the present invention, including, e.g., cholesterolconjugation, as such modification has been found to significantlyimprove in vivo pharmacological properties (Soutschek J, et al. Nature2004 432: 173-78).

RNA may be produced enzymatically or by partial/total organic synthesis,any modified nibonucleotide can be introduced by in vitro enzymatic ororganic synthesis. In one embodiment, an RNAi agent is preparedchemically. Methods of synthesizing RNA molecules are known in the art,in particular, the chemical synthesis methods as de scribed in Verma andEckstein (1998) Annul Rev. Biochem. 67:99-134. In another embodiment, anRNAi agent (e.g. a siRNA) is prepared enzymatically. For example, ads-siRNA can be prepared by enzymatic processing of a long ds RNA havingsufficient complementarity to the desired target mRNA. Processing oflong ds RNA can be accomplished in vitro, for example, using appropriatecellular lysates and ds-siRNAs can be subsequently purified by gelelectrophoresis or gel filtration. ds-siRNA can then be denaturedaccording to art-recognized methodologies. In an exemplary embodiment,RNA can be purified from a mixture by extraction with a solvent orresin, precipitation, electrophoresis, chromatography, or a combinationthereof. Alternatively, the RNA may be used with no or a minimum ofpurification to avoid losses due to sample processing. Alternatively,the siRNA can also be prepared by enzymatic transcription from syntheticDNA templates or from DNA plasmids isolated from recombinant bacteria.Typically, phage RNA polymerases are used such as T7, T3 or SP6 RNApolymerase (Milligan and Uhlenbeck (1989) Methods Enzymol. 180:51-62).The RNA may be dried for storage or dissolved in an aqueous solution.The solution may contain buffers or salts to inhibit annealing, and/orpromote stabilization of the single strands.

The instant invention additionally provides for delivery of shRNAshaving enhanced specificity or efficacy in mediating RNAi. In contrastto short siRNA duplexes, short hairpin RNAs (shRNAs) mimic the naturalprecursors of miRNAs and enter at the top of the RNAi pathway. For thisreason, shRNAs are believed to mediate RNAi more efficiently by beingfed through the entire natural RNAi pathway.

shRNAs have a single strand that forms a duplex stem including twoportions that are generally complementary, and a loop, that connects thetwo portions of the stem. In a preferred embodiment, short hairpin RNAsof the invention are artificial constructs engineered to deliver desiredsiRNAs.

In shRNAs employed in certain embodiments of the instant invention, oneportion of the duplex stem is a nucleic acid sequence that iscomplementary (or anti-sense) to the target mRNA. Thus, shRNAs include aduplex stem with two portions and a loop connecting the two stemportions. The two stem portions are about 18 or 19 to about 25, 30, 35,37, 38, 39, or 40 or more nucleotides in length. When used in mammaliancells, the length of the stem portions should be less than about 30nucleotides to avoid provoking non-specific responses like theinterferon pathway. In non-mammalian cells, the stem can be longer than30 nucleotides. In fact, the stem can include much larger sectionscomplementary to the target mRNA (up to, and including the entire mRNA).The two portions of the duplex stem must be sufficiently complementaryto hybridize to form the duplex stem. Thus, the two portions can be, butneed not be, fully or perfectly complementary. In addition, the two stemportions can be the same length, or one portion can include an overhangof 1, 2, 3, or 4 micleotides. The overhanging nucleotides can include,for example, uracils (Us), e.g., all Us. The loop in the shRNAs can be2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides inlength.

shRNAs of the invention include the sequences of the desired siRNAduplex. The desired siRNA duplex, and thus both of the two stem portionsin the shRNA, are selected by methods known in the art.

The shRNAs of the invention can be synthesized by standard methods knownin the art, e.g., by use of an automated DNA synthesizer (such as arecommercially available from Biosearch, Applied Biosystems, etc.). TheshRNAs can be used directly as described below or cloned into expressioncassettes or vectors by methods known in the field.

Such cassettes or vectors can be constructed by recombinant DNAtechnology methods known in the art. Vectors can be plasmid, viral, orother vectors known in the art such as those described herein, used forreplication and expression in mammalian cells or other targeted celltypes. The nucleic acid sequences encoding the shRNAs of the inventioncan be prepared using known techniques. For example, two synthetic DNAoligonucleotides can be synthesized to create a novel gene encoding theentire shRNA. The DNA oligonucleotides, which will pair, leavingappropriate ‘sticky ends’ for cloning, can be inserted into arestriction site in a plasmid that contains a promoter sequence (e.g., aPol II or a Pol III promoter) and appropriate terminator sequences 3′ tothe shRNA sequences (e.g., a cleavage and polyadenylation signalsequence from SV40 or a Pol III terminator sequence).

The invention also encompasses cells that contain any of the foregoingexpression vectors and thereby express the nucleic acid molecules of theinvention in the cell. The cells can be cultured using known techniquesand methods (see, e.g., Culture of Animal Cells (R. I. Freshney, Alan R.Liss, Inc. 1987); Molecular Cloning, Sambrook et al. (Cold Spring HarborLaboratory Press, 1989)).

The expression of the shRNAs of certain embodiments of the invention isdriven by regulatory sequences, and the vectors of the invention caninclude any regulatory sequences known in the art to act in mammaliancells. The term regulatory sequence includes promoters, enhancers, andother expression control elements. A person skilled in the art would beable to choose the appropriate regulatory sequence.

The regulatory sequences can be inducible or constitutive. Suitableconstitutive regulatory sequences include the regulatory sequence of ahousekeeping gene such as the α-actin regulatory sequence, or may be ofviral origin such as regulatory sequences derived from mouse mammarytumor virus (MMTV) or cytomegalovirus (CMV).

Alternatively, the regulatory sequence can be selected to directexpression in specific organs or cell types (see, e.g., Lasko et al.,1992, Proc. Natl. Acad. Sci. USA 89:6232). Several tissue-specificregulatory sequences are known in the art including the albuminregulatory sequence for liver (Pinkert et al., 1987, Genes Dev.1:268276); the endothelin regulatory sequence for endothelial cells(Lee, 1990, J. Biol. Chem. 265:10446-50); the keratin regulatorysequence for epidermis; the myosin light chain-2 regulatory sequence forheart (Lee et al., 1992, J. Biol. Chem. 267:15875-85), and the insulinregulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl.Acad. Sci. USA 83:2511-2515), or the vav regulatory sequence forhematopoietic cells (Oligvy et al., 1999, Proc. Natl. Acad. Sci. USA96:14943-14948). Another suitable regulatory sequence, which directsconstitutive expression of transgenes in cells of hematopoietic origin,is the murine MHC class I regulatory sequence (Morello et al., 1986,EMBO J. 5:1877-1882). Since NMC expression is induced by cytokines,expression of a test gene operably linked to this regulatory sequencecan be upregulated in the presence of cytokines.

In addition, expression of a transgene encoding an shRNA can beprecisely regulated, for example, by using an inducible regulatorysequence and expression systems such as a regulatory sequence that issensitive to certain physiological regulators, e.g., circulating glucoselevels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Suchinducible expression systems, suitable for the control of transgeneexpression in cells or in mammals such as mice, include regulation byecdysone, by estrogen, progesterone, tetracycline, chemical inducers ofdimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG)(collectively referred to as “the regulatory molecule’). Each of theseexpression systems is well described in the literature and permitsexpression of the transgene throughout the animal in a manner controlledby the presence or absence of the regulatory molecule. Such expressionsystems can be employed, e.g., during transplantation of tissues oftransgenic animals. For a review of inducible expression systems, see,e.g., Mills, 2001, Genes Devel. 15:1461-1467, and references citedtherein.

The regulatory elements referred to above include, but are not limitedto, the cytomegalovirus hCMV immediate early gene, the early or latepromoters of SV40 adenovirus (Bemoist et al., Nature, 290:304, 1981),the tet system, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage A, the controlregions of fd coat protein, the promoter for 3-phosphoglycerate kinase,the promoters of acid phosphatase, and the promoters of the yeasta/α-mating factors. Additional promoters include the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al.,Cell 22:787-797, 1988); the herpes thymidine kinase promoter (Wagner etal., Proc. Natl. Acad. Sci. USA 78:1441, 1981); or the regulatorysequences of the metallothionein gene (Brinster et al., Nature 296:39,1988).

RNAi agents of the invention (e.g., siRNAs, shRNAs) can be synthesizedin vivo, in situ, or in vitro. Endogenous RNA polymerase of the cell maymediate transcription in vivo or in situ, or cloned RNA polymerase canbe used for transcription in vivo or in vitro. For transcription from atransgene in vivo or an expression construct, a regulatory region (e.g.,promoter, enhancer, silencer, splice donor and acceptor,polyadenylation) may be used to transcribe the RNAi agent (e.g. asiRNA). Inhibition may be targeted by specific transcription in anorgan, tissue, or cell type; stimulation of an environmental condition(e.g., infection, stress, temperature, chemical inducers); and/orengineering transcription at a developmental stage or age. A transgenicorganism that expresses an RNAi agent (e.g. a siRNA) from a recombinantconstruct may be produced by introducing the construct into a zygote, anembryonic stem cell, or another multipotent cell derived from theappropriate organism.

RNAi agents and the constructs encoding the RNAi agents of the instantinvention can also be delivered to transplanted tissues by employingadenovirus and other art-recognized systems for gene therapy. Suchsystems can be implemented with the methods, tissues and compositions ofthe instant invention to deliver RNAi agents, e.g., to humans.

III. RNAi Targets

The present invention features methods and compositions for themanipulation of gene expression in transplantable cells and/or tissues.In certain embodiments, the invention features the delivery of siRNAboth in vivo and in vitro to silence expression of one or more targetgenes. The compositions and methods of the invention are particularlysuited for altering the phenotype of transplantable cells and/ortissues. For example, manipulation of gene expression levels at the timeof cell and/or tissue procurement and/or isolation could serve toenhance the survival and function of the cells and/or tissue and couldresult in greater clinical success in further transplantation of saidcells or tissues. Moreover, siRNA is a non-viral method of altering geneexpression and thus would be preferred for immunosuppressed transplantpatients.

In certain embodiments, the invention features delivery of siRNA duringor through the procurement of an organ (e.g., via administration via thevasculature of said organ) and during or through isolation oftransplantable cells from said organ. siRNA-mediated gene therapy can beapplied in several instances during the organ procurement process: 1)pre-procurement via intravenous perfusion in the deceased donor; 2) viaorgan perfusion prior to packaging for transport; and/or 3) in cellculture, for example, in the case of islet transplantation. The methodsand compositions of the invention may therefore be applied in anycombination with procurement of an organ and/or tissue, e.g., siRNA maybe used to alter phenotype prior to procurement of an organ or tissueand/or siRNA may be used to alter phenotype of an organ or tissuefollowing procurement.

The methods and compositions of the invention may be applied to anyorgan and/or tissue to change the phenotype of that organ and/or tissue.For example, the methods and compositions of the invention may beapplied to treatment of cytomegalovirus (CMV) in transplantable tissue,e.g., to alter virus replication or otherwise inhibit propagation ofthis virus, which is commonly a latent infection found in transplantableorgans and/or tissues. The methods and compositions of the invention mayalso be used, e.g., to treat or prevent hepatitis C in liver tissuesprior to and/or during transplantation, as transmission of hepatitis Cpresents a common and important problem for liver transplantation.

The methods and compositions of the invention may also be applied duringoncology treatments, e.g., during surgical oncology. For example, RNAiagents may be administered using the methods of the invention forpurpose of inhibiting replication of aberrant (e.g., tumor) cells.Inhibition of cell cycle genes and other such genes vital to tumorpropagation can enhance oncology treatments.

In preferred aspects of the invention, the target mRNA molecule of theinvention specifies the amino acid sequence of a protein associated witha deleterious outcome of organ transplantation, which most commonly caninclude proteins associated with organ rejection, proteins associatedwith a viral infection and/or pathological condition, and proteinsassociated with the process of apoptosis/cell death.

The MHC genes may be targeted in combatting tissue rejection. Inpreferred embodiments of the instant invention, the gene targeted by theRNAi agent encodes for an MHC molecule characteristic of the graftedtissue. For MHC class II genes, any of the genes of the DP, DQ and DRsubregions for class II α chain and β chain genes (i.e., DPα, DPβ, DQα,DQβ, DRα, and DRβ) can be targeted. Similarly, in certain embodiments ofthe instant invention, MHC class I genes are targeted, including any ofthe genes in the HLA-A, HLA-B or HLA-C subregions for class I α chaingenes, or β₂-microglobulin (located outside the MHC complex on aseparate chromosome in humans) may also be targeted. Prevention of organrejection may also be achieved by approaches comprising direct targetingof genes encoding for proteins associated with T cell activity and ofgenes encoding for proteins associated with appropriate expression,processing, trafficking and/or delivery/cellular localization of, e.g.,MHC gene products and T cell gene products associated with organrejection. In addition, adhesion molecules (e.g., CTLA-4) may also betargeted to inhibit tissue rejection by the methods and compositions ofthe instant invention.

In certain embodiments of the invention, the target gene(s) encode for aprotein that may be a pathogen-associated protein (e.g., a viral proteininvolved in immunosuppression of the host, replication of the pathogen,transmission of the pathogen, or maintenance of the infection), or ahost protein which facilitates entry of the pathogen into the host, drugmetabolism by the pathogen or host, replication or integration of thepathogen's genome, establishment or spread of infection in the host, orassembly of the next generation of pathogen. In such embodiments, theinvention features a method for treating, stabilizing, or preventing aninfection in a subject (e.g., a vertebrate such as a mammal or human) orcell, tissue or organ derived from such a subject. These methods involveintroducing into the subject or cell, tissue or organ derived therefroman RNAi agent. The RNAi agent has substantial sequence identity and/oris substantially complementary to a region of a target gene in apathogen (e.g., a virus, (e.g., a herpesvirus (e.g., herpes simplex,varicella-zoster virus, Epstein-Barr virus, cytomegalovirus (CMV)),hepatitis C, HIV, JC virus), bacteria or yeast), or in a cell, tissue ororgan infected with a pathogen and specifically inhibits expression ofthe target gene. The pathogen can be an intracellular or extracellularpathogen, and the target gene can be, e.g., a gene of the pathogen thatis necessary for replication and/or pathogenesis, or a gene encoding fora cellular receptor necessary for a cell, tissue or organ to be infectedwith the pathogen.

In a preferred aspect of the instant invention, a gene(s) encoding for aprotein involved in apoptosis is targeted. Such genes include, e.g.,bcl-2, p53, caspases, cytotoxic cytokines such as TNF-α or Fas ligand,and a number of other genes art-recognized as capable of mediatingapoptosis. In certain embodiments of the invention, genes involved incell growth can also be directedly targeted by the RNAi agents of theinvention. Such genes include oncogenes (e.g., genes encoding for ABLI,BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI,ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2. MLL, MYB,MYC. MYCLI, MYCN, NRAS, PIM I. PML, RET, SRC, TALI, TCL3, and YES), aswell as genes encoding for tumor suppressor proteins (e.g., APC, BRCA1,BRCA2, MADH4, MCC, NF I, NF2, RB I., TP53, and WTI).

Alternatively, the gene targeted by the RNAi agent(s) of the instantinvention can encode for a protein that may be a tumor-associatedprotein or an autoimmune disease-associated protein.

In exemplary embodiments, siRNA is delivered during or through theprocurement of a pancreas (e.g., a donor pancreas) as well as during orthrough isolation of pancreatic islets from the pancreas. For deliveryduring the procurement of a pancreas, administration of the siRNA to theendocrine microvasculature is preferable. In certain embodiments, theinvention features delivery of siRNA during or through the procurementof an organ (e.g., via administration via the vasculature of thepancreas) and during or through isolation of transplantable cells (e.g.,islets) from the pancreas. siRNA-mediated gene therapy can be applied inseveral instances during procurement of the pancreas: 1) pre-procurementvia intravenous perfusion in the deceased donor; 2) via organ perfusionprior to packaging the pancreas for transport; and/or 3) in pancreaticcell (e.g., islet cell) culture. The compositions and methods of theinvention are particularly suited for altering the phenotype ofpancreatic (e.g., islet) cells. In preferred applications, thecompositions and methods of the invention are useful for altering genessuch that islet engraftment, viability and/or function is enhanced,leading to improved outcomes in islet transplantation.

In certain embodiments, in vitro delivery (e.g., transfection) of siRNA(e.g., transfection) is such that at least a 25% reduction in targetmRNA levels is observed. For example, siRNA is delivered at aconcentration, dosage or according to a dosage regime (e.g., multipledoses over set time interval) such that the percent reduction in targetmRNA levels is achieved. In other embodiments, in vitro delivery ofsiRNA is such that at least a 30-35% reduction in target mRNA levels isobserved. In other embodiments, in vitro delivery of siRNA is such thatat least a 35-40% reduction in target mRNA levels is observed. In otherembodiments, in vitro delivery of siRNA is such that at least a 40-45%reduction in target mRNA levels is observed. In other embodiments, invitro delivery of siRNA is such that at least a 45-50% reduction intarget mRNA levels is observed. In yet other embodiments, in vivodelivery of siRNA is such that at least a 55%, 60%, 70% or 80% reductionin target mRNA levels is observed.

In certain embodiments, in vitro delivery (e.g., transfection) of siRNA(e.g., transfection) is such that at least 30% of the target cellsincorporate the siRNA. In other embodiments, in vitro delivery (e.g.,transfection) of siRNA (e.g., transfection) is such that at least 35% ofthe target cells incorporate the siRNA. In other embodiments, in vitrodelivery (e.g., transfection) of siRNA (e.g., transfection) is such thatat least 40% of the target cells incorporate the siRNA. In yet otherembodiments, in vitro delivery (e.g., transfection) of siRNA (e.g.,transfection) is such that at least 45%, 50%, 55% or 60% of the targetcells incorporate the siRNA.

In exemplary embodiments, about 50-100 nM siRNA is delivered to orcontacted with the target cells. In further embodiments, about 100-200nM siRNA is delivered to or contacted with the target cells. In furtherembodiments, about 200-300 nM siRNA is delivered to or contacted withthe target cells. In further embodiments, about 300-400 nM siRNA isdelivered to or contacted with the target cells. In still furtherembodiments, greater than 500 nM siRNA is delivered to or contacted withthe target cells.

IV. Methods of Introducing RNAs and RNAi Agents

Physical methods of introducing nucleic acids include injection of asolution containing the nucleic acid (e.g., RNAi agent), bombardment byparticles covered by the nucleic acid (e.g., RNAi agent), soaking thecell, tissue, allograft or organism in a solution of the nucleic acid(e.g., RNAi agent), or electroporation of cell membranes ortissues/allografts in the presence of the nucleic acid (e.g., RNAiagent). Lipid-mediated carrier transport (e.g., liposome-mediateddelivery) of an RNAi agent to a cell, tissue, allograft or organism ispreferred in certain embodiments of the instant invention. A viralconstruct packaged into a viral particle may also accomplish bothefficient introduction of an expression construct and/or RNAi agent intothe cell, tissue, allograft or organism of the instant invention; and inthe instance of a virally introduced expression construct, transcriptionof an RNAi agent may then occur. Other methods known in the art forintroducing nucleic acids to cells may be used, such aschemical-mediated transport, such as calcium phosphate, and the like.Thus the nucleic acid (e.g., RNAi agent) may be introduced along withcomponents that perform one or more of the following activities: enhancenucleic acid (e.g., RNAi agent) uptake by the cell, inhibit annealing ofsingle strands, stabilize the single strands, or other-wise increaseinhibition of the target gene.

The nucleic acid (e.g., RNAi agent) may be directly introduced into thecell (i.e., intracellularly), tissue, organ, allograft or organism; orintroduced extracellularly into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing a cell, tissue, organ, allograft or organism in a solutioncontaining the nucleic acid (e.g., RNAi agent). The bile or biliarysystem, vascular or extravascular circulation, the blood or lymphsystem, and the cerebrospinal fluid are sites where the nucleic acid(e.g., RNAi agent) may be introduced.

In certain embodiments of the invention, an RNAi agent is exposed to atransplanted tissue (e.g. an organ) by perfusion. A tissue containing avascular system is procured from a donor subject by methods adhering toclinical protocols, using cold storage in an appropriate solution (e.g.,UW solution, also referred to as Belzer Organ Preservation solution).The isolated tissue may be tested to determine whether the RNAi agentcan be perfused into the exocrine/endocrine tissues. “Catheters” areinserted into the tissue for input and output using the vascular system.Tubing of the “catheters” is connected to a peristaltic pump and thetissue is submerged in a chamber containing preservation solution (e.g.UW solution) at 4° C. Preservation solution, optionally containing atracking dye (e.g., 5% trypan blue) then perfuses the tissue. The RNAiagent, optionally labeled with a fluorescent dye (e.g., Cy3), is alsoadded to the perfusion media and allowed to circulate. While beingperfused with the RNAi agent, the tissue may also be submitted toelectric pulses via an electroporator to further diffuse the RNAi agentinto the tissue and optimize delivery in situ. Following perfusiontreatment, the tissue may be transplanted. Alternatively, if the tissueis to be used for research purposes, the tissue may be perfused for24-48 hours, with the tissue then removed, fixed in buffered formalin,paraffin embedded, and sectioned. Fluorescence microscopy may then beused to identify the extent to which the RNAi agent has been deliveredto the perfused tissue (in instances of pancreatic perfusion, e.g.,labeled anti-insulin antibodies may be used to identify islet cells,with these islet cells additionally observed for delivery of the RNAiagent, in determining the extent to which the RNAi agent has beendelivered to the exocrine and endocrine tissues).

Depending on the particular target gene and the dose of RNAi agentdelivered, this process may provide partial or complete loss of functionfor the target gene. A reduction or loss of gene expression in at least50%, 60%, 70%, 80%, 90%, 95% or 99% or more of targeted cells isexemplary. Inhibition of gene expression refers to the absence (orobservable decrease) in the level of protein and/or mRNA product from atarget gene. Specificity refers to the ability to inhibit the targetgene without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS).

In certain embodiments, in vivo delivery of siRNA is such that at leasta 20% reduction in target mRNA levels is observed. For example, siRNA isdelivered at a concentration, dosage or according to a dosage regime(e.g., multiple doses over set time interval) such that the percentreduction in target mRNA levels is achieved. In other embodiments, invivo delivery of siRNA is such that at least a 25-30%/o reduction intarget mRNA levels is observed. In other embodiments, in vivo deliveryof siRNA is such that at least a 30-35% reduction in target mRNA levelsis observed. In yet other embodiments, in vivo delivery of siRNA is suchthat at least a 40%, 50%, 60% or 70% reduction in target mRNA levels isobserved.

Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a sample, e.g., a cell,tissue or organ not treated according to the present invention. Lowerdoses of injected material and longer times after administration of aRNAi agent may result in a controlled decrease in inhibition achieved.Quantitation of gene expression in a cell, tissue or organ may showsimilar amounts of inhibition at the level of accumulation of targetmRNA or translation of target protein. As an example, the efficiency ofinhibition may be determined by assessing the amount of gene product inthe cell; mRNA may be detected with a hybridization probe having anucleotide sequence outside the region used for the inhibitorydouble-stranded RNA, or translated polypeptide may be detected with anantibody raised against the polypeptide sequence of that region.

The RNAi agent may be introduced in an amount which allows delivery ofat least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500or 1000 copies per cell) of material may yield more effectiveinhibition; lower doses may also be useful for specific applications.

V. Delivery Vehicles for RNAi Agents

The RNAi agent(s) of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,polymers, receptor targeted molecules, or other formulations, forassisting in uptake, distribution and/or absorption. The subject RNAiagent(s) can be provided in formulations also including penetrationenhancers, carrier compounds and/or transfection agents.

The RNAi agents of the invention also encompass any pharmaceuticallyacceptable salts, esters or salts of such esters, or any other compoundwhich, upon administration to a cell, tissue or organ, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto RNAi agents and pharmaceutically acceptable salts of the siRNAs,pharmaceutically acceptable salts of such RNAi agents, and otherbioequivalents.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,NI-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, eraylenediamine. N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids.

For siRNA molecules, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid tannic acid,palmitic acid alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonicacid, polygalacturonic acid, and the like; and (d) salts formed fromelemental anions such as chlorine, bromine, and iodine.

A. Supramolecular Complexes

The subject RNAi agent(s) of the instant invention may be formulated aspart of a “supramolecular complex.” To further illustrate, the RNAiagent can be contacted with at least one polymer to form a composite andthen the polymer of the composite treated under conditions sufficient toform a supramolecular complex containing the RNAi agent and amulti-dimensional polymer network. The polymer molecule may be linear orbranched. Accordingly, a group of two or more polymer molecules may belinear, branched, or a mixture of linear and branched polymers. Thecomposite may be prepared by any suitable means known in the art. Forexample, the composite may be formed by simply contacting, mixing ordispersing the RNAi agent with a polymer. A composite may also beprepared by polymerizing monomers, which may be the same or different,capable of forming a linear or branched polymer in the presence of theexpression agent. The composite may be further modified with at leastone ligand, e.g., to direct cellular uptake of the expression agent orotherwise effect tissue or cellular distribution in vivo of theexpression agent. The composite may take any suitable form and,preferably, is in the form of particles.

B. Liposome Formulations

In certain preferred embodiments, the invention provides compositionincluding dsRNA or dsRNA-encoding plasmids that are encapsulated orotherwise associated with liposomes. Packaging and/or encapsulation ofRNAi agents of the invention in liposomes may increase the efficiency ofdelivery of such agents. Merely to illustrate, dsRNA moieties ordsRNA-encoding plasmids can be condensed with a polycationic condensingagent, suspended in a low-ionic strength aqueous medium, and cationicliposomes formed of a cationic vesicle-forming lipid. The ratio ofliposome lipids to plasmid can be adjusted achieving maximumtransfection. That ratio, in mole liposome lipid/μg plasmid, will oftenbe greater than 5 but less than 25, and preferably greater than 8 butless than 18, and more preferably greater than 10 but less than 15 andmost preferably between 12-14. Such complexes preferably have asubstantially homogeneous size (i.e., ±20%, preferably ±10% or morepreferably ±5% in size) of typically less than about 200 nm andpreferably in the range of 50-200 nm.

Liposomes, as used herein, refer to lipid vesicles having an outer lipidshell, typically formed on one or more lipid bilayers, encapsulating anaqueous interior. In certain embodiments, the liposomes are cationicliposomes composed of between about 20-80 mole percent of a cationicvesicle-forming lipid, with the remainder neutral vesicle-forming lipidsand/or other components. As used herein, “vesicle-forming lipid” refersto any amphipathic lipid having hydrophobic and polar head groupmoieties and which by itself can form spontaneously into bilayervesicles in water, as exemplified by phospholipids. A preferredvesicle-forming lipid is a diacyl-chain lipid, such as a phospholipid,whose acyl chains are typically between about 14-22 carbon atoms inlength, and have varying degrees of unsaturation.

A cationic vesicle-forming lipid is one whose polar head group with anet positive charge, at the operational pH, e.g., pH 4-9. Typicalexamples include phospholipids, such as phosphatidylethanolamine, whosepolar head groups are derivatized with a positive moiety, e.g., lysine,as illustrated, for example, for the lipid DOPE derivatized withL-lysine (LYS-DOPE) (Guo, et al., 1993). Also included in this class arethe glycolipids, such as cerebrosides and gangliosides having a cationicpolar head-group.

Another cationic vesicle-forming lipid which may be employed ischolesterol amine and related cationic sterols. Exemplary cationiclipids include 1,2-diolelyloxy-3-(trimethylanuno) propane (DOTAP);N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromide (DMRIE); N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3β[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Choi); anddimethyldioctadecylammonium (DDAB).

The remainder of the liposomes are formed of neutral vesicle-forminglipids, meaning vesicle forming lipids which have no net charge or whichmay include a small percentage of lipids having a negative charge in thepolar head group. Included in this class of lipids are thephospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM),and cholesterol, cholesterol derivatives, and other uncharged sterols.

The above-described lipids can be obtained commercially, or preparedaccording to published methods. Other lipids that can be included in theinvention are glycolipids, such as cerebrosides and gangliosides.

The RNAi agent-liposome complex employed in certain embodiments of theinstant invention may include liposomes having a surface coating ofhydrophilic polymer chains, effective to extend the blood circulationtime of the plasmid/liposome complexes. Suitable hydrophilic polymersinclude cyclodextrin (CD), polyethylene glycol (PEG), polylactic acid,polyglycolic acid, polyvinyl-pyrrolid-one, polymethyloxazoline,polyethyloxazoline, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatized celluloses,such as hydroxymethylcellulose or hydroxyethyl-cellulose. A preferredhydrophilic polymer chain is polyethyleneglycol (PEG), preferably as aPEG chain having a molecular weight between 500-10,000 daltons, morepreferably between 1,000-5,000 daltons. The hydrophilic polymer may havesolubility in water and in a non-aqueous solvent, such as chloroform.

The coating is preferably prepared by including in the vesicle-forminglipids a phospholipid or other diacyl-chain lipid, derivatized at itshead group with the polymer chain. Exemplary methods of preparing suchlipids, and forming polymer coated liposomes therewith, have beendescribed in U.S. Pat. Nos. 5,013,556, and 5,395,619, which areincorporated herein by reference.

It will be appreciated that the hydrophilic polymer can be stablycoupled to the lipid, or coupled through an unstable linkage whichallows the polymer-coated plasmid-liposome complexes to shed or“release” the hydrophilic polymer coating during circulation in thebloodstream or after localization at a target site. Attachment ofhydrophilic polymers, in particular polyethyleneglycol (PEG), tovesicle-forming lipids through a bond effective to release the polymerchains in response to a stimulus have been described, for example in WO98/16202, WO 98/16201, which are hereby incorporated by reference, andby Kirpotin, D. et a. (FEBS Left. 1996 388, 115-118).

The releasable linkage, in one embodiment, is a chemically releasablelinkage which is cleaved by administration of a suitable releasing agentor is cleaved under selective physiological conditions, such as in thepresence of enzymes or reducing agents. For example, ester and peptidelinkages are cleaved by esterase or peptidase enzymes. Disulfidelinkages are cleaved by administration of a reducing agent, such asglutathione or ascorbate, or by a reducing agent present in vivo, suchas cysteine, which is present in plasma and intracellularly.

Other releasable linkages include pH sensitive bonds and bonds which arecleaved upon exposure to glucose, light or heat. By way of an example,the hydrophilic polymer chains can be attached to the liposome by a pHsensitive bond, and the plasmid-liposome complexes are targeted to asite having a pH effective to cleave the bond and release thehydrophilic chains, such as a tumor region. Exemplary pH sensitive bondsinclude acyloxyalkyl-ether, acetal and ketal bonds.

Another example is where the cleavable bond is a disulfide bond, broadlyintended herein to refer to sulfur-containing bonds. Sulfur-containingbonds can be synthesized to achieve a selected degree of lability andinclude disulfide bonds, mixed sulfide-sulfone bonds andsulfide-sulfoxide bonds. Of the three bonds, the disulfide bond is leastsusceptible to thiolysis and the sulfide-sulfoxide bond is mostsusceptible.

Such releasable bonds are useful to tailor the rate of release of thehydrophilic polymer segment from the liposome complexes. For example, avery labile disulfide bond can be used for targeting to blood cells,isolated islet cells or endothelial cells, since these cells are readilyaccessible and a shorter liposome blood circulation or delivery lifetimeis sufficient. At the other extreme, a long-lasting or hearty disulfidebond can be used when the target is a whole organ or other tissue wherea longer liposome blood circulation or delivery lifetime is generallyneeded for the complexes to reach the desired target.

The releasable bond attaching the hydrophilic polymer chains to theliposome is cleaved in vivo typically as a result of change inenvironment, such as when the liposomes reach a specific site with aslightly lower pH, such as a region of tumor tissue, or a site withreducing conditions, such as a hypoxic tumor. Reducing conditions invivo can also be effected by administration of a reducing agent, such asascorbate, cysteine or glutathione. The cleavable bond may also bebroken in response to an external stimulus, such as light or heat.

Additionally, the liposome complexes may include an affinity moiety ortargeting ligand effective to bind specifically to target cells at whichthe therapy is aimed. Such moieties can be attached to the surface ofthe liposome or to the distal ends of hydrophilic polymer chains.Exemplary moieties include antibodies, ligands for specific binding totarget cell surface receptors and the like, as described, for example,in PCT application Nos. WO US94/03103, WO 98/16202 and WO 98/16201. Themoiety can also be a hydrophobic segment to facilitate fusion of thecomplex with a target cell.

Polycationic condensing agents used to condense the dsRNA anddsRNA-encoding plasmids can be multiply charged cationic polymers, andare preferably biopolymers such as such as spermidine, spermine,polylysine, protamine, total histone, specific histone fractions such asH1, H2, H3, H4, and other polycationic polypeptides, but may alsoinclude biocompatible polymers, such as polymyxin B. It will beappreciated that these polycationic condensing agents can be used infree base or salt forms, for example, protamine sulfate and polylysinehydrobromide. The polycationic condensing agent may be a histone, which,as referred to herein, includes total histone or specific histonefractions.

The hydrophobic segment in the polymer-lipid conjugate may be ahydrophobic polypeptide sequence. Preferably, the polypeptide sequenceconsists of about 5-80, more preferably 10-50, most preferably 20-30,non-polar and/or aliphatic/aromatic amino acid residues. These sequencesare active in triggering fusion of certain enveloped viruses with hostcells and include Parainfluenza viruses, such as Sendai, Simian Virus-5(SV5), measles virus, Newcastle Disease Virus (NDV) and RespiratorySyncytial Virus (RSV)—Other examples include human retroviruses, such asHuman Immunodiffiency Virus-1 (HTV-1), the causative agent of AIDS,which infects cells by fusion of the virus envelope with the plasmamembrane of the host cell. Fusion occurs at physiological (i.e.,neutral) pH and is followed by injection of the viral genetic material(nucleocapsid) into the cytoplasmic compartment of the host cell.

C. Ligand-Directed Formulations

The supramolecular complexes and liposomes of the subject invention canbe associated with one or more ligands effective to bind to specificcell surface proteins or matrix on the target cell, thereby facilitatingsequestration of the complex to target cells, and in some instances,enhancing uptake of the RNAi agent by the cell. Merely to illustrate,examples of ligands suitable for use in targeting the supramolecularcomplexes and liposomes of the present invention to specific cell typesare listed in Table 1 below.

TABLE 1 Sample ligands of potential use in targeting liposomes and otherRNAi agent delivery complexes Ligand Receptor Cell type folate fblatereceptor epithelial carcinomas, bone marrow stem cells water solublevitamins vitamin receptor various cells pyridoxyl phosphate CD4 CD4⁺lymphocytes apolipoproteins LDL Liver hepatocytes, vascular endothelialcells insulin insulin receptor transferrin transferrin receptorendothelial cells galactose asialoglycoprotein liver hepatocytesreceptor sialyl-Lewis_(x) E, P selectin activated endothelial cellsMac-1 L selectin neutrophils, leukocytes VEGF Flk-1,2 tumor epithelialcells basic FGF FGF receptor tumor epithelial cells EGF EGF receptorepithelial cells VCAM-1 a₄b₁ integrin vascular endothelial cells ICAM-1a_(L)b₂ integrin vascular endothelial cells PECAM-1/CD31 a_(v)b₃integrin vascular endothelial cells, activated platelets osteopontina_(v)b₁ integrin endothelial cells and a_(v)b₅ integrin smooth musclecells in atherosclerotic plaques RGD sequences a_(v)b₃ integrin tumorendothelial cells, vascular smooth muscle cells HIV GP 120/41 or CD4CD4⁺ lymphocytes GP120

VI. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted target geneexpression or activity. “Treatment”, or “treating” as used herein, isdefined as the application or administration of a therapeutic agent(e.g., an RNAi agent, vector or transgene encoding same) to a patient,or application or administration of a therapeutic agent to an isolatedtissue or cell line from a patient, who has a disease or disorder, asymptom of disease or disorder or a predisposition toward a disease ordisorder, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve or affect the disease or disorder, thesymptoms of the disease or disorder, or the predisposition towarddisease.

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target genemolecules of the present invention or target gene modulators accordingto that individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

In one aspect, the invention provides a method for preventing in asubject, tissue rejection, disease or other deleterious transplantationoutcome which is caused or contributed to by aberrant or unwanted targetgene expression or activity, by administering to the subject atherapeutic agent (e.g., an RNAi agent, e.g., an RNAi modulatory agent,or vector or transgene encoding same). Subjects at risk for suchdeleterious transplantation outcome(s) can be identified by, forexample, any or a combination of diagnostic or prognostic assays asdescribed herein. Prophylactic treatment can occur prior to themanifestation of symptoms characteristic of the target gene aberrancy,such that a disease, disorder, or outcome is prevented or,alternatively, delayed in its progression.

Another aspect of the invention pertains to methods for treating asubject having or experiencing tissue rejection, disease or otherdeleterious transplantation outcome which is caused or contributed to byaberrant or unwanted target gene expression or activity. Accordingly, inan exemplary embodiment, the modulatory method of the invention involvescontacting a cell, tissue or organ capable of expressing target genewith a therapeutic agent (e.g., an RNAi agent, e.g., an RNAi modulatoryagent, or vector or transgene encoding same) that is specific for thetarget gene or protein such that expression or one or more of theactivities of target protein is modulated. These modulatory methods canbe performed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder (e.g., transplantrejection) characterized by aberrant or unwanted expression or activityof a target gene polypeptide or nucleic acid molecule. Inhibition oftarget gene activity is desirable in situations in which target gene isabnormally unregulated and/or in which decreased target gene activity islikely to have a beneficial effect.

Therapeutic methods and compositions can be tested in an appropriateanimal model. For example, a modulatory agent or RNAi agent (orexpression vector or transgene encoding same) as described herein can beused in an animal model to determine the efficacy, toxicity, or sideeffects of treatment with said agent. Alternatively, a therapeutic agentcan be used in an animal model to determine the mechanism of action ofsuch an agent.

While the methods of the instant invention primarily involve treatmentof transplanted animal tissues with RNAi agents, such RNAi agents mayalso be administered to transplanted plant tissues. Administration of anRNAi agent to a transplanted plant tissue may be performed through,e.g., injection of the RNAi agent to a plant or plant tissue in a mannerequivalent to those methods of the instant invention related toperfusion of animal tissues. Compositions designed to aid administrationof agents to plants are described, for example, in WO 03/020024, thecontents of which are incorporated herein by reference.

VII. Pharmaceutical Compositions

The invention pertains to uses of the above-described agents fortherapeutic treatments as described infra. Accordingly, the modulatorsor agents of the present invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the RNAi agent and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation),transdermal (topical), and transmucosal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components; a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

In certain embodiments of the instant invention. RNAi agents areprepared with carriers that will protect the compound against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems. The RNAiagents of the invention may also be prepared with carriers that willenhance delivery and/or targeting of the agents to an appropriate cell,tissue or organ. Such carriers and delivery vehicles are art-recognized,with certain preferred delivery modes for the instant invention detailedabove.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The invention is further described in the Examples below which describethe successful treatment of commonly transplanted cells and whole organswith RNAi agents. The following examples should not be construed aslimiting. The contents of all references, patents and published patentapplications cited throughout this application are incorporated hereinby reference.

EXAMPLES Example I: Effective Ex Vivo Treatment of Isolated Islet Cellswith siRNA

The efficacy of administration of siRNA to islet cells in culture wasexamined. Islet cells were isolated from C57BL/6 mice by methodspreviously described (Ricordi, C and Rastellini, C. Automation in IsletIsolation. In: Ricordi, C, ed. Methods in Cell Transplantation. Austin,Tx, USA: RG Landes Co. 1995: Salvalaggio PRO et al. Transplantation.2002, 74:877-900), and these cells were employed to assess the abilityof siRNA molecules directed against a green fluorescent protein (GFP) todownregulate production of GFP expressed from an exogenously introducedadenoviral vector (adeno-GFP). Successful cellular incorporation of theanti-GFP siRNA was anticipated to diminish green fluorescence ofcultured islet cells. Three groups were used: 1) a negative control,wherein cells were administered no siRNA, 2) a non-specific siRNAcontrol, and 3) siRNA directed to inhibit GFP (anti-GFP siRNA). Thoseislet cells transfected with siRNA were transfected for 24 hours,allowing enough time for absorbed siRNA to prime RISC prior tosubsequent infection of the islet cells with adeno-GFP for 24 hours.Reduced levels of green fluorescence were observed for islet cells thatwere treated with anti-GFP siRNA, as compared to fluorescence observedfor those cells either left untreated or treated with a non-specificsiRNA (FIG. 1). An siRNA dose of 100 nM using a Lipofectamine 2000(Invitrogen) transfection kit was observed to effect introduction of thesiRNA into 20 islet cells per culture well. Anti-GFP siRNA was thereforeshown to effectively reduce GFP production in cultured islet cells.

Example II: Effective Perfusion of Whole Pancreas In Situ with siRNA

Given the efficacy observed for siRNA administration to cultured isletcells, studies were performed to examine the effect of siRNA perfusionto whole pancreas. Initial studies were conducted to test surgicalprocedures for siRNA injection into pancreas of BALB/c mice, siRNA waseither injected as “naked” molecules or packaged into liposomes usingLipofectamine 2000 (Invitrogen). The control siRNA used was labeled withthe red-fluorophore, Cy-3, to track the siRNA location in the tissue. 1c.c. containing 2 μg siRNA/liposomes in UW (University of Wisconsin,Belzer Organ Preservation) solution+6% trypan blue was injected viaretrograde portal vein. siRNA injections with naked siRNA resulted inpoor diffusion into the tissue. After injections, pancreata were removedand stored at 4° C. for 20 hours. Organs were fixed in bufferedformalin, paraffin embedded, and sections were stained for insulin withimmunofluorescence. The fluorescent images of insulin-stained pancreaticislets from siRNA-treated versus untreated mice pancreata are shown inFIG. 2. Both siRNA-treated sections showed substantial siRNA uptake bythe islets as observed in the Cy-3 red fluorescent images, when comparedto the background auto-fluorescence in the untreated control section(compare FIG. 2, center panels A and B with center panel C). Thus,successful direct organ perfusion with siRNA was demonstrated.

Example III: Suppressed Insulin Production Via Whole Pancreas siRNAPerfusion

Whole mouse pancreas was perfused (by the methods of the precedingExample) with anti-Insulin 2 siRNA (Ambion pre-designed siRNA ID#62419). Such experiments demonstrated the efficacy with which perfusedRNAi agents were capable of suppressing production of a nativetranscript in a whole tissue. Following perfusion of whole pancreas withanti-Insulin 2 siRNA, islet cells were isolated using standard isletpreparation procedures as described, e.g., in Jonas, J. C., et al.(Diabetes 1998 47, 1266-73). Isolated islet cells were then transferredto wells for performance of real-time RT-PCR. Each well contained 200islet cells. Real-time RT-PCR was performed in triplicate on lysed cellsusing a FAM-labeled Insulin-specific TaqMan probe (Roche MolecularSystems, Inc.), with FAM fluorescence detected by an ABI 5700 machine.Islet cells isolated from whole mouse pancreas that had been perfusedwith varying concentrations (25 nM, 50 nM, 100 nM, and 200 nM) ofanti-Insulin 2 siRNA exhibited dramatic reductions in insulinproduction, when compared to control islet cells isolated from pancreasthat had been perfused with a control (anti-luciferase) siRNA. Wholetissue-perfused RNAi agents were thus shown to dramatically reduceproduction of a native transcript.

Example IV: Perfusion of Transplantable Tissues

Demonstration of successful direct organ perfusion with siRNA enablesthe improvement of transplantation outcomes through contacting tissueswith siRNA prior to or during transplantation. siRNA may be directlyperfused during or shortly after the organ procurement process. Asorgans are dissected and isolated on their vascular pedicles,organ-specific siRNA can be delivered into the tissue via any afferent(or efferent retrograde) vessel.

Example V: Effective Delivery and Function of siRNA in Isolated IsletsIn Vitro

Pancreatic islets were isolated from BALB/c mice, and 200 islets wereplaced into wells of a 48 well plate in 1 ml 10% FBS CMRL 1066supplemented Cellgro media (Mediatech, Inc., Hemdon, Va.). Samples weredivided into 3 groups: (a) untreated control. (b) Ins2-siRNAtransfected, and (c) Luc-siRNA transfected islets as a non-specificsiRNA control. For in vitro delivery, the siRNA were packaged usingLipofectamine 2000 (Invitrogen, Carlsbad, Calif.) at a DNA-to-liposome(μg/μl) ratio of 1:3, following the manufacturer's recommendations in adelivery volume of 100 μl. Dose titrations of 0, 50, 100, 200, and 400nM final concentration were used for the experimental and non-specificcontrol siRNA. Samples were transfected and incubated at 37° C., 5% CO₂for 48 hours followed by RNA isolation for cDNA synthesis andquantitative-RT-PCR. To determine to what extent siRNA could transfectpancreatic islets, fluorescent conjugated siRNAs were used following thesame protocol used in the dose titration. A Cy-3-labeled Luc-siRNA wasused to qualitatively assess the incorporation of siRNA utilizingfluorescent microscopy and FACS was used to quantitate siRNAtransfection efficiency using a FITC-labeled Luc-siRNA.

FIG. 4A demonstrates that primary islets were transfected with theCy-3-siRNA and clearly shows that siRNA can be introduced into islets inculture. FACS data in FIG. 4B, revealed that more than 45% of isletcells incorporated the FITC-labeled siRNA (400 nM siRNA concentration).FIG. 4C depicts the normalized real-time PCR data comparing the Ins2expression levels of samples from the three groups and demonstrates thatall concentrations of Ins2-siRNA used were able to significantly reduceIns2 transcript levels in pancreatic islets, in vitro. In sum, it wasshown that Ins2-siRNA can functionally inhibit Ins2 gene expression inpancreatic islets when delivered in vitro by transfection.

Example VI: Effective Delivery and Function of siRNA in PancreaticIslets in Vivo by Tail Vein Injection

For hydrodynamic injections synthetic siRNA, 100 μg in 0.8 ml PBS, wasrapidly injected into one of the side tail veins. The siRNA wasdelivered ‘naked’, without liposomal transfection reagents. To examinein vivo siRNA cellular uptake and transfection efficiency in pancreaticislets, siRNA-treated and PBS treated control mice (n=3) were used. Onehundred μg of Cy-3-Luc-siRNA, a red fluorescent fluorophore, wasadministered to 3 mice of the siRNA-treated group. Islets were isolatedand purified four hours later from all mice in both groups and werecultured for 16 hours using 48 well plates in 1 ml 10% FBS CMRL 1066supplemented Cellgro media, followed by imaging with fluorescentmicroscopy. Following the same protocol using a FITC-Luc-siRNA, isolatedislets were treated with 0.25% typsin/EDTA then prepared for FACSanalysis to quantitatively determine the percentage of islet cells thatincorporated FITC-Luc-siRNA. To assess Ins2-siRNA function in pancreaticislets, mice were divided into 3 groups (n=3/group) as follows: group 1,the PBS-treated control; group 2, received Ins2-siRNA: group 3, was anon-specific siRNA control receiving Luc-siRNA. Four hours after micereceived tail vein injections their islets were isolated and culturedfor 16 hours (37° C. 5% CO₂) followed by total RNA isolation for cDNAsynthesis and quantitative RT-PCR for measuring the Ins2 gene transcriptlevels. All experiments were carried out in triplicate.

Fluorescent microscope images (FIG. 5A) revealed all diffusely redislets from the mice that received Cy-3-siRNA while no red fluorescencewas observed from the control treated islets. These images showed thatsiRNA was able to reach the islets by way of the pancreatic endocrinemicrovasculature, and also showed that in vivo delivery of siRNA topancreatic islets was also achievable. The measurement of siRNAtransfection efficiency using FITC-Luc (FIG. 5B) revealed that 19% ofislet cells had incorporated siRNA. Assessment of Ins2-siRNA function invivo is shown in FIG. 5C as a bar graph of the quantitative RT-PCRnormalized data. The Ins2 expression level from the treated controlsample was set at 100%. The average value from Ins2-siRNA treatedsamples showed a significant reduction in expression compared tountreated control samples (65.5% vs. 34.5%; P<0.05). The siRNA effectwas gene specific as the non-specific siRNA controls had the same Ins2expression levels as PBS treated control. Thus, it was shown thatIns2-siRNA can functionally inhibit Ins2 gene expression in pancreaticislets when delivered by intravenous injection in vivo.

Example VII: Exemplary siRNA Formulations

The methods of the present invention can be applied using anyart-recognized siRNA. Exemplary siRNA molecules, including twonewly-derived siRNA molecules that target the viral DNA polymerase ofhuman cytomegalovirus (HCMV), that can be used in practicing the presentinvention are shown in Table 2.

TABLE 2Examples of siRNA Sequences for Targeting of Sample Candidate Hostand Viral Genes (SEQ ID NOS 1-11, respectively, in order of appearance)Human Gene Target siRNA Sequence Reference PML-4 5′AACGACAGCCCAGAAGAGGAA JBC 279:1838-44, 2004 c-myc 5′ CAGAAAUGUCCUGCAAUUUBreast Cancer Res. 7:220-228, 2005 Bcl-2 5′ GUGAAGUCAACAUGCCUGC-dTdTClin Cancer Res. 10:7721-6, 2005 BAX 5′ UAUGGAGCUGCAGAGGAUGdTdTCancer Res. 65:309-16, 2005 BID 5′ GAAGACAUCAUCCGGAAUA-dTdTJBC 279:35047-52, 2004 Caspase 3 5′ UGGAUUAUCCUGAGAUGGGdTdTBlood 103:4310-6, 2004 Caspase 8 5′ CUACCAGAAAGGUAUACCUdTdTNature 419:395-9, 2002 APaf-1 5′ GUGAACCAGGAUGGGUCACCAScience 297:1352-54, 2002 H-ras 5′ GGGCAAGAGUGCGCUGACCAUCOncogene 22:5694-5701, 2003 Viral (HCMV) Gene Target siRNA SequenceReference UL54 5′ CUGCUCAACAAGUGGGUUU-dTdT New UL54 5′GUUUUCAGAGCCGUGUUUU-dTdT New

Thus, Table 2 is exemplary of siRNAs that can be used in the methods ofthe instant invention; and one of skill in the art will recognize thatthe methods of the invention can be practiced with any number of siRNAmolecules exerting preventive and/or therapeutic effects intransplantation.

The use of gene therapy in transplant research has mainly focused on again of function strategy where the gene of interest is expressed usinga viral vector for delivery. Animal studies have explored attempts toreduce graft rejection by over-expressing genes whose products willinterfere in the co-stimulatory cascade preventing donor T cellactivation. Genes used have encoded the fusion proteins CTLA-4Ig andCD40-Ig, expressed from adenovirus (Takehara, M et al. Human Gene Ther.2001 12: 415-426; Guillot, C et al. J Immunol. 2002 168: 1600-1609).Induction of molecular chimerism by expression of virally transducedallogeneic donor-type MHC class I genes in bone marrow has had limitedsuccess leading to the prolonged survival of cardiac and skin allografts(Tian, C et al. Front Biosci. 2002 7: 1331-1337; Bagley. J et al. Blood.2002 99: 4394-4399). However, although viral vectors are promising inbasic science and pre-clinical models, safety concerns have arisen dueto fatalities reported from clinical gene therapy trials using viralvectors (Thomas, C E et al. Nature Rev Genetics 2003 4: 346-358) andtheir use in transplant is potentially dangerous because of recipientimmunosuppression. In contrast, RNAi offers a non-viral method for genemodulation and thus can potentially eliminate the complicationsassociated with viral vectors, while the utilization of siRNA adopts astrategy of loss of gene function.

RNA interference of gene expression is an attractive method for alteringa phenotype of transplantable tissue. For example, manipulation of geneexpression levels at the time of islet procurement and isolation canserve to enhance the survival and function of the islet cell mass andcould then theoretically result in greater clinical success in islettransplantation. In the present examples, it is demonstrated thatIns2-siRNA can functionally inhibit Ins2 gene expression in pancreaticislets whether delivered in vitro by transfection or by intravenousinjection in vivo. Quantitative RT-PCR confirms the functionaltransfection of siRNA for an organ-specific gene, thus evidencingproof-of-concept.

It was hypothesized that isolated intact islets could be sufficientlytransfected with siRNA which would significantly reduce target geneexpression. The FACS and quantitative RT-PCR data (FIG. 4) validatedthis hypothesis. This reduction was specific in its effect, as controlsiRNA did not significantly reduce Ins2 beyond background levels. Thefunctional data demonstrates that siRNA can silence genes in primarywhole islets, in vitro, also both the fluorescent images for the siRNAdose titration and the more quantitative FACS data help document howmuch siRNA was incorporated into islet cells. It is postulated thatcationic liposomal formulations may increases siRNA transfectionefficiency in vitro. The transfection efficiency of 46% withLipofectamine 2000 in primary islets indicates that siRNA may penetratebeyond the periphery into a larger percentage of an islet cell mass thanpreviously thought. The above examples demonstrate that transfectionefficiency is preserved through the procurement of an organ andisolation of potentially transplantable cells (islets). These datademonstrate both the in vivo delivery and functional inhibition oftarget gene expression by siRNA targeted to an endogenous gene inpancreatic islets. Both the fluorescent tagged siRNA experiments showedincorporation of siRNA into pancreatic islets after in vivoadministration. The FACS data showed that 19% of the islet cells insuspension had incorporated the synthetic siRNA molecules, this lowefficiency may be due to the physical transport of siRNA across the cellmembrane, as well as, a dosage effect. The real-time PCR data revealedthat tail vein administration of 100 μg Ins2-siRNA significantly(P<0.05) reduced the Ins2 gene transcript levels. It has been shown byfluorescent imaging that in vivo delivery of ‘naked’ siRNA to pancreaticislets is feasible with intravenous tail vein injection without suchhigh pressures as to damage vessels or harm the animal. Microvascularstudies of the dense pancreatic islet capillary network describe aphysical route existing through the porous capillary lumen basementmembrane and fenestrated endothelium through which siRNA molecules maycontact individual islet cells, and especially β-cells. These datasupport the observation that transfection of the siRNA in vivo does notrequire a liposomal based transfection reagent, indicating that nakedsiRNA may have an endocytic pathway of entry into cells. Interestingly,it has been demonstrated that intravenous administration of siRNAminimally induces, if any, an IFN response in mice (Heidel, J D et al.Nat Biotechnol. 2004 22: 1579-1582). It is proposed that the ability todeliver siRNA to the donor pancreatic islets prior to procurement andisolation may allow the gene silencing process to initiate and have agreater effect on phenotypic change.

What is claimed:
 1. A method of treating a subject at risk of or havinga disorder associated with apoptosis-induced ischemic injury in anorgan, the method comprising administering to the subject an RNAi agentcapable of downmodulating ischemia-induced apoptosis, wherein the RNAiagent comprises a sense strand and an anti-sense strand, wherein theanti-sense strand is sufficiently complementary to p53 RNA to reduce theproduction of p53 via RNA-induced silencing complex (RISC) so as tocounter apoptosis-induced ischemic injury in the organ, and wherein saidadministering comprises injecting the RNAi agent.
 2. The method of claim1, wherein the organ is a kidney or liver.
 3. The method of claim 1,wherein the RNAi agent is in a preservation solution that compriseseither saline or an immunosuppressant.
 4. The method of claim 1, whereinthe RNAi agent is an siRNA molecule.
 5. The method of claim 1, whereinthe RNAi agent is an shRNA molecule.
 6. The method of claim 1, whereinthe RNAi agent comprises an oligonucleotide comprising a modification toa 2′-OH group, the modification being selected from the group consistingof the 2′-OH group replaced by a H, alkoxy or OR, halogen, SH, SR,amino, or CN group, wherein R is a lower alkyl, alkenyl, alkynyl, oraryl.
 7. The method of claim 6, wherein the RNAi agent comprises a2′-O-methyl oligonucleotide.
 8. The method of claim 1, wherein the RNAiagent contacts the organ via lipid-mediated delivery or as a componentof a liposomal delivery preparation.